Monte Carlo simulations of the relative biological effectiveness for DNA double strand breaks from 300 MeV u(-1) carbon-ion beams.

Monte Carlo simulations are used to calculate the relative biological effectiveness (RBE) of 300 MeV u(-1) carbon-ion beams at different depths in a cylindrical water phantom of 10 cm radius and 30 cm long. RBE values for the induction of DNA double strand breaks (DSB), a biological endpoint closely related to cell inactivation, are estimated for monoenergetic and energy-modulated carbon ion beams. Individual contributions to the RBE from primary ions and secondary nuclear fragments are simulated separately. These simulations are based on a multi-scale modelling approach by first applying the FLUKA (version 2011.2.17) transport code to estimate the absorbed doses and fluence energy spectra, then using the MCDS (version 3.10A) damage code for DSB yields. The approach is efficient since it separates the non-stochastic dosimetry problem from the stochastic DNA damage problem. The MCDS code predicts the major trends of the DSB yields from detailed track structure simulations. It is found that, as depth is increasing, RBE values increase slowly from the entrance depth to the plateau region and change substantially in the Bragg peak region. RBE values reach their maxima at the distal edge of the Bragg peak. Beyond this edge, contributions to RBE are entirely from nuclear fragments. Maximum RBE values at the distal edges of the Bragg peak and the spread-out Bragg peak are, respectively, 3.0 and 2.8. The present approach has the flexibility to weight RBE contributions from different DSB classes, i.e. DSB0, DSB+ and DSB++.

Inhibition of plant vacuolar H(+)-ATPase by diethylpyrocarbonate.

Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.

A lysine residue involved in the inhibition of vacuolar H(+)-pyrophosphatase by fluorescein 5'-isothiocyanate.

Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PP(i) hydrolysis. A fluorescent probe, fluorescein 5'-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H(+)-PPase. The enzymatic activity and its associated H(+) translocation of vacuolar H(+)-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H(+)-PPase. Changes in K(m) but not V(max) values of vacuolar H(+)-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H(+)-PPase could be protected by its physiological substrate, Mg(2+)-PP(i). These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H(+)-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region.

Comparative study on the ALA photodynamic effects of human glioma and meningioma cells.

BACKGROUND AND OBJECTIVE: The purpose of this study was to compare the differential susceptibility to photodynamic therapy (PDT) mediated damage in human U-105MG glioma cells and CH-157MN meningioma cells in vitro using 5-amino-levulinic acid (ALA) as photosensitizer, and to determine if growth factors would enhance PDT-mediated damage of these cells. STUDY DESIGN/MATERIALS AND METHODS: U-105MG or CH-157MN cells were irradiated with polychromatic light in the presence of ALA. A Xenon lamp (150 W) was used as the light source. For the study on the effect of growth factor on ALA-PDT, cells were cultured in serum free medium for 24 hours. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet derived growth factor BB (PDGF-BB) was added to achieve a final concentration of 50 ng/ml. 30 minutes later, cells were incubated with ALA (100 microg/ml) for 24 hours, washed, and irradiated with light (11 J/cm2). MTT tetrazolium assays were performed 24 hours after light irradiation. RESULTS: The inhibition of metabolic cellular function in U-105MG cells by ALA depended on both light energy density and ALA concentration. The susceptibility to ALA-PDT was profoundly lower for CH-157MN meningioma cells than U-105MG glioma cells. When incubated with ALA (100 microg/ml), U-105MG cells exhibited an LD50 around 8 J/cm2 of light irradiation, whereas that of CH-157MN cells was more than 25 J/cm2. EGF, bFGF, or PDGF-BB did not have any effects on the susceptibility of these two cell lines to ALA-PDT. CONCLUSION: ALA-PDT was more effective in killing U-105MG glioma cells than CH-157MN meningioma cells. The differential susceptibility was likely due to differential accumulation of PpIX in these cells. EGF, bFGF, or PDGF-BB did not have stimulatory or inhibitory effect on the efficiency of ALA-PDT.

Regulation of vascular endothelial growth factor secretion in human meningioma cells.

Previously, we induced vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) secretion in glioma cell lines by using physiologic concentrations of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet-derived growth factor-BB (PDGF-BB). We hypothesized that VEGF/VPF might enhance the blood supply required for the unregulated growth of tumors, and that it acts as the central mediator of tumor angiogenesis. The objective of this study was to determine whether the expression of VEGF/VPF by meningiomas is regulated by growth factors or sex hormones. By means of an enzyme-linked immunosorbent assay of CH-157MN meningioma cell supernatants, we demonstrated that EGF and bFGF similarly induce VEGF secretion by CH-157MN meningioma cells. At the maximum concentrations of EGF (50 ng/mL) and bFGF (50 ng/mL) used in this study, VEGF secretion was induced to 140% to 160% above baseline constitutive secretion. PDGF-BB homodimer did not enhance VEGF secretion significantly. Estradiol (up to 10(-7) mol/L), progesterone (up to 10(-5) mol/L), or testosterone (up to 10(-5) mol/L) did not stimulate or inhibit VEGF secretion in CH-157MN meningioma cells (p > 0.05). Furthermore, we demonstrated that dexamethasone decreased VEGF secretion to 32% of baseline constitutive secretion. This might explain the effect of corticosteroids in alleviating peritumoral brain edema in meningiomas. These results suggest that VEGF secretion in CH-157MN meningioma cells is mainly regulated by growth factors and corticosteroids, but not by sex hormones. Understanding the regulation of VEGF/VPF secretion in meningiomas might contribute to the development of a new therapeutic strategy.

Mutational analysis of the conserved cationic residues of Bacillus stearothermophilus 6-phosphoglucose isomerase.

The importance in catalysis of the conserved arginine (R207) and lysine residues (K144, K294, K356, and K425) of 6-phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site-directed mutagenesis and kinetic analysis. In general mutations had minor effects on the Km for fructose 6-phosphate. More dramatic effects were seen on kcat. The R207A mutant had a five orders of magnitude decrease in kcat relative to the wild-type enzyme. There was a significant recovery, by three orders of magnitude, in the kcat for the R207K mutant. The results suggest that the positive charge provided by R207 plays a critical role in the isomerization reaction. K425 was substituted with alanine, valine, phenylalanine, tryptophan and aspartate. All mutant enzymes at position 425 had kcat decreased in the range of several-hundred-fold. For the other mutants, K294A and K144A, the kcat values were 3.5% and 27% of the wild-type enzyme, respectively. No effects on catalysis were observed for the K356A mutant. The results suggest that R207, K144, K294, and K425 are located in the active site of the enzyme. The active-site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5'-phosphate on enzymatic activities. The data also confirm the importance of K425 and K144 anticipated by the affinity labeling studies of the corresponding residues by pyridoxal 5'-phosphate in pig muscle phosphoglucose isomerase.

Expression, purification, and crystallization of two isozymes of 6-phosphoglucose isomerase of Bacillus stearothermophilus.

Two isozymes of 6-phosphoglucose isomerase (phosphoglucose isomerase A & phosphoglucose isomerase B), isolated from Bacillus stearothermophilus, have been overexpressed in Escherichia coli strain DF2145 and purified to homogeneity. Crystals of both isozymes have been obtained by the vapor diffusion method. The crystals of phosphoglucose isomerase A have unit cell dimensions a = b = 132.0 A, c = 183.6 A, and diffract to about 2.8 A resolution. An analysis of the reflection data indicates that the crystal system is hexagonal, space group P6122 or P6522. The crystals of phosphoglucose isomerase B complexed with 6-phosphogluconate belong to the orthorhombic space group I222 (or I212121), with cell dimensions a = 75.1 A, b = 95.7 A, c = 171.5 A, and diffract to a resolution of 2.3 A. These crystals promise to yield more detail for the substrate recognition and higher resolution structures of 6-phosphoglucose isomerase.

Palmar hyperhidrosis in children: treatment with video endoscopic laser sympathectomy.

Palmar hyperhidrosis (PH) often starts in childhood and can be a disabling condition for a significant number of young children at the age they begin primary school. There are few reports regarding the surgical treatment of PH in children. The authors report on 40 PH patients under 16 years of age treated with video thoracoscopic laser sympathectomy; there has been substantial experience with this procedure for the treatment of adults with PH. A satisfactory result, with very low morbidity, was achieved for all 40 children. The surgical technique is described briefly. With the technique, the proper sympathetic segment is visualized in almost all cases and then definitely ablated with a fiberoptic low-power laser while under the aid of sympathetic monitoring. Consequently, an adequate sympathectomy warranting a long-lasting therapeutic effect can be achieved without the need of tissue diagnosis. No case required conversion to open sympathectomy. Neither injury to the lung nor bleeding was encountered. Horner's syndrome did not occur in any case. Bilateral sympathectomy was accomplished generally within 30 minutes. All patients were discharged after an overnight stay and are doing well with normal activities. The most frequent complication was compensatory hyperhidrosis, which was tolerable after reassurance. Based on the accumulated experience, it is justified to recommend early surgery, with this refined technique, in cases of severe PH in children.

Autonomic activities in hyperhidrosis patients before, during, and after endoscopic laser sympathectomy.

Three hundred palmar hyperhidrosis (PH) patients have been treated with video endoscopic laser sympathectomy during the last 2 years. Monitoring the palmar skin perfusion (PSP) and palmar skin temperature (PST) has been used intraoperatively to aid the confirmation of the correct sympathetic segment for laser ablation. The preoperative and postoperative PSP and PST and sympathetic skin response (SSR) also have been measured to evaluate the therapeutic effect of this method. An apparent increase of PSP would occur intraoperatively after the interruption of the T2 sympathetic segment, and then a gradual elevation of PST would follow after the extirpation of the segment. A rise of PST of about 3 degrees C after laser ablation of the appropriate segment indicated sufficient denervation of the hand and predicted long-lasting relief of PH. Furthermore, both PSP and PST also significantly increased after the operation. The postoperative elevation of the PST (usually about 3 degrees C) is similar to that recorded during intraoperative monitoring. The amplitude and the latency of SSR in the palm and sole were recorded both before and after sympathectomy. A remarkable decrease of palmar SSR amplitude and its ratio was found postoperatively by comparing it with that of plantar SSR in the same patient. These autonomic activity changes have correlated well with the postoperative satisfaction of the patients. Based on our study, the anatomic identification confirmed by the sympathetic monitorings has proved essential to achieve a definite and adequate sympathectomy leading to a satisfactory resolution of PH without the need of a tissue diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Video thoracoscopic laser sympathectomy for palmar hyperhidrosis.

Palmar hyperhidrosis (PH) is common in Orientals from subtropical areas. Many therapeutic modalities are used in practice, but none has proved to be entirely satisfactory. We have developed a new therapeutic technique by combining a video thoracoscopic system with a surgical laser unit (both waveguide CO2 laser and fibre-optic Nd:YAG laser). The operation was performed under general anaesthesia with alternative one-lung ventilation. With this technique, we are able to identify the sympathetic trunk on the TV screen and confirm its proper level with accurate ablation by intraoperative vasomotor monitoring. Consequently, an adequate sympathectomy can be definitely achieved through laser extirpation. We have successfully treated 300 PH patients with this technique from 1990 to 1992. The ages ranged from six to 63 years with a mean of 26.6. There were 125 males and 175 females. Most patients underwent en bloc ablation of the T2 segment which includes a major part of the T2 ganglion with its adjacent trunk which overlays the T2 rib head. All of them obtained a satisfactory relief of PH except 13 patients. The procedure did not result in a change of vital signs. There was neither obvious injury to lung nor bleeding. No Horner's syndrome was produced. The commonest complication was compensatory hyperhidrosis in various degrees encountered in about half of the cases. Two-thirds of the patients were followed up for more than 12 months and only three had recurrence. Based on our experience, the technique is considered to be a minor and safe procedure and able to achieve a definite and long-lasting therapeutic effect. It causes minimal discomfort and scarring. Particularly, the operation time and hospital stay were markedly shortened in comparison with other conventional open sympathectomy procedures.

Fluorospectral study of the rat brain and glioma in vivo.

An animal model of cerebral glioma was utilized by implanting C6 glioma cells into the brains of adult Wistar rats. Once tumors developed to 7-12 mm in diameter, we conducted continuous fluorimetry monitoring of glioma up to 24 hours using a fibre-optic system connected to an intensified multichannel photodetector after an intravenous injection of hematoporphyrin derivative (HPD) into the rats. The intensity of the fluorescence in normal brain reached a plateau 6 hours after intravenous injection of HPD while that in glioma reached a plateau 80 minutes after injection. These fluorescence intensities of glioma, brain adjacent to tumor (BAT), and surrounding normal brain were measured in vivo 24 hours after intravenous administration of 5 mg/kg of HPD. The ratio of fluorescence intensities between glioma and brain was 6.1 while the ratio between BAT and brain was 3.9. There were no obvious differences in shapes between the spectra of the natural fluorescence (autofluorescence) of rat glioma and brain but the intensity of autofluorescence was much weaker in glioma. There are many problems in spectroscopic studies of biological tissues in vivo. It cannot be overemphasized that very strict criteria must be applied in order to get accurate data. Fluorescence from HPD administration may be used to discriminate tumor tissue from surrounding normal brain tissue during operation if the measuring conditions could be kept constant. It is important to understand the photospectral properties of glioma and brain tissue in order to get the most benefits in clinical application of light-induced fluorescence or photoradiation therapy.

Antitumor activity of tetraacetylglucosamine mustard.

1,3,4,6-Tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose is active against L1210 leukemia, giving over 100% increased life-span at optimal dose. Against P388 leukemia, it gives 200% increased life-span with long-term survivors. The compound is most active when given i.p., but shows some activity when given s.c. than p.o., and is more potent (therapeutic and toxic effect) than mechlorethamine on both a molar and a mg basis. Of importance, the schedule dependency for the administration of 1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose in L1210 leukemia differs from most alkylating agents in that it is best given by multiple daily injections rather than as a single large injection on Day 1. This characteristic can be attributed to the amino-glucose moiety.

Synthfsis of bis(aziridinyl)phosphinyl-N-hydroxyurethane derivatives as antineoplastic agents.

Several new "dual antagonists" were synthesized in which the 2,2-dimethyl (or ring C unsubstituted) aziridine phosphinyl function is linked to N-hydroxyurethane rather than the urethane moiety. Three of the new compounds showed very high activities against leukemia L1210 in mice.