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PubMed2000

Selective boosting of tumor subvolumes.

PURPOSE AND BACKGROUND: It is no longer considered mandatory to deliver a uniform dose to the tumor volume in radiotherapy. Non-uniform doses are unavoidable in brachytherapy and in stereotactic radiosurgery, with often good results. Deliberately non-uniform doses may increase tumor control probability (TCP) and enable steeper dose gradients outside the treated volume to be achieved. New methods of tumor imaging might show regions of specific activity or hypoxia which could be selectively targeted. This paper investigates by modeling the effect of boosting, by dose ratios up to 2, for a range of tumor subvolumes. METHODS AND MATERIALS: A standard linear-quadratic algorithm was used to define the dose-response curve for tumors of various volumes (numbers of clonogenic cells), radiosensitivity (SF(2)), assumed slope (gamma(50)) and dose for 50% tumor control (TCD(50)). Curves of tumor control probability (TCP) were constructed to show the increase of TCP, as a function of the ratio of boost dose to the TCD(50), above the baseline 50% TCP, for a set of different proportions of tumor volume boosted. RESULTS: Calculated values of TCP increased rapidly with both boost dose ratio and with proportion of volume boosted. The increase in TCP reached a plateau after boost dose ratios of 1.2-1.3, as has been noted before, except where very large proportions of tumor volume exceeding 90% were boosted. Quite large increases of TCP, to about 75%, could be achieved if the gamma(50) slope was steep, and especially in small tumors (having fewer cells). Radiosensitivity was not an independent factor because radiosensitive tumors had a low TCD(50) and this was the baseline dose considered as unity. CONCLUSION: There were few situations where a boost dose ratio exceeding 1.3 appeared to be worthwhile or necessary. Significant increases of TCP, up from 50% to 75%, might therefore be achieved for a small increase in risk of necrosis, where a substantial proportion of tumor volume (60-80%) could be boosted.

Algorithms↗
PubMed2001

Synthesis and characterization of alkanethiolate-coordinated iron porphyrins and their dioxygen adducts as models for the active center of cytochrome p450: direct evidence for hydrogen bonding to bound dioxygen.

Two kinds of novel cytochrome P450 models, which have alkanethiolate axial ligands and hydroxyl groups inside molecular cavities, were designed and synthesized as functional O(2) binding systems. A superstructured porphyrin, designated as "twin-coronet" porphyrin, was used as the common framework of the model complexes. This porphyrin bears four binaphthalene bridges on the both sides and forms two pockets surrounded by the bulky aromatic rings. Thiobenzyloxy and thioglycolate moieties, which contain an alkanethiolate group exhibiting various electron-donating abilities and degrees of bulkiness, were covalently linked to twin-coronet porphyrin to yield thiolate-coordinated hemes, TCP-TB and TCP-TG (twin-coronet porphyrin with thiobenzyloxy and thioglycolate groups), respectively. Both ferric complexes exhibited high stability during usual experimental manipulation under air and were characterized by MS, UV/vis, ESR spectroscopies, and CV. The ESR spectra exhibited low-spin signals (TCP-TB: g = 2.334, 2.210, 1.959; TCP-TG: g = 2.313, 2.209, 1.966). The cyclic voltammogram of TCP-TB in CH(3)CN gave a quasi-reversible wave which corresponds to the Fe(III)/Fe(II) redox couple: E(p)()(/2) = -1.35 V (vs Fc/Fc(+)). On the other hand, TCP-TG showed a fine reversible wave: E(1/2) (Fe(III)/Fe(II)) = -1.12 V. The stable dioxygen adducts were formed in the reaction of the ferric complexes with KO(2) under an oxygen atmosphere and characterized by UV/vis and resonance Raman (RR) spectroscopies. In the RR spectra, the nu(O--O) bands of the dioxygen adducts were observed at 1138 cm(-1) (TCP-TB) and 1137 cm(-1) (TCP-TG). The hypothesis that hydrogen bonding between the bound oxygen and the hydroxyl groups of the binaphthyl moieties could increase their stability was verified by RR spectroscopy. When all hydroxyl groups were deuterated, only the frequencies of the nu(O--O) bands were upshifted by 2 cm(-1) without any perturbation in the porphyrin skeleton. This work shows the first direct evidence for a hydrogen bond to dioxygen in an oxy form of a thiolate-coordinated heme model system. These results are discussed in context of the process of dioxygen binding and activation in cytochrome P450.

Cytochrome P-450 Enzyme System↗
PubMed2005

Sorption of 2,4,6-trichlorophenol in model humic acid-clay systems.

Humic acids and clays are important soil components that influence the sorption and desorption of organic contaminants; however, it is unclear how humic acids influence the sorption of organic contaminants onto clays and their subsequent desorption. Sorption and desorption of 2,4,6-trichlorophenol (2,4,6-TCP) by and from humic acid-modified K(+)- and Ca(2+)-montmorillonite and -illite were compared with unmodified clays using batch equilibration methods. Commercial humic acid and the humic acid extracted from forest soil were employed in this experiment. The adsorbed amount of 2,4,6-TCP by commercial humic acid was almost twice as large as that adsorbed by the extracted soil humic acid. More 2,4,6-TCP was sorbed onto K(+)- and Ca(2+)-illite than onto K(+)- and Ca(2+)-montmorillonite. K(+) clays were more effective in adsorbing 2,4,6-TCP than Ca(2+) clays. Sorption of 2,4,6-TCP on humic acid-modified Ca(2+)- and K(+)-montmorillonite and -illite increased as compared with unmodified clays. The sorption nonlinearity of 2,4,6-TCP on humic acid-modified Ca(2+)- and K(+)-illite increased remarkably as compared with the unmodified clays. The sorption nonlinearity of 2,4,6-TCP on humic acid-modified Ca(2+)- and K(+)-montmorillonite increased slightly in contrast to unmodified montmorillonites. By comparing sorption and desorption results, we observed hysteresis for all sorbents including humic acids, clays, and humic acid-modified clays. Sorption nonlinearity and hysteresis were dependent on the structure of humic acids. Higher aromaticity of humic acids resulted in greater sorption nonlinearity and desorption hysteresis. In addition, sorption capacity (K(f)') was positively correlated with the humic acid content of the sorbents. These results show that modification of humic acids on clays can not only increase the adsorption ability of clays but also affect the sorption nonlinearity of 2,4,6-TCP, and the desorption hysteresis was probably due to the structural characteristics of humic acids.

Adsorption↗
PubMed1994

Density of NMDA-coupled and uncoupled 1-[1-(2-[3H]thienyl) cyclohexyl]piperidine recognition sites in the brain and spinal cord: differential effects of NMDA agonists and antagonists.

Binding of 1-[1-(2-[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or sigma recognition sites. In both tissues, [3H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [3H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [3H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [3H]TCP sites may be associated with sigma binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 nM [3H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [3H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [3H]TCP-labeled binding sites in the spinal cord may be associated with sigma but not the NMDA receptor-coupled PCP sites.

Animals↗
PubMed2003

Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37.

AIMS: To analyse the possible effect of poly-beta-hydroxyalkanoate (PHA) consumption on 2,4,6-trichlorophenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP. METHODS AND RESULTS: The strain was inoculated in saline solution supplemented with 2,4,6-TCP (25-400 microm). Chlorophenol degradation was followed both spectrophotometrically and by chlorine released; viable bacterial counts were also determined. Cells starved for 24, 48 or 72 h were incubated with 25 microm of 2,4,6-TCP and PHA in cells investigated by spectrofluorimetric and flow cytometry. Results demonstrated that starvation decreased the ability to degrade 2,4,6-TCP. After 72 h of starvation, degradation of 2,4,6-TCP decreased to less than 10% and the relative PHA content diminished to ca 50% during the first 24 h. CONCLUSION: Utilization of PHA may be an important factor for the degradation of toxic compounds, such as 2,4,6-TCP, in bacterial strains unable to use this toxic compound as carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing a relationship between intracellular PHA consumption and 2,4,6-TCP degradation. Therefore, PHAs provides an endogenous carbon and energy source under starvation and can play a significant role in the degradation of toxic compounds.

Alphaproteobacteria↗
PubMed1998

TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

Amino Acid Sequence↗
PubMed2006

Optimization of tumour control probability in hypoxic tumours by radiation dose redistribution: a modelling study.

Tumour hypoxia is a known cause of clinical resistance to radiation therapy. The purpose of this work was to model the effects on tumour control probability (TCP) of selectively boosting the dose to hypoxic regions in a tumour, while keeping the mean tumour dose constant. A tumour model with a continuous oxygen distribution, incorporating pO(2) histograms published for head and neck patients, was developed. Temporal and spatial variations in the oxygen distribution, non-uniform cell density and cell proliferation during treatment were included in the tumour modelling. Non-uniform dose prescriptions were made based on a segmentation of the tumours into four compartments. The main findings were: (1) Dose redistribution considerably improved TCP for all tumours. (2) The effect on TCP depended on the degree of reoxygenation during treatment, with a maximum relative increase in TCP for tumours with poor or no reoxygenation. (3) Acute hypoxia reduced TCP moderately, while underdosing chronic hypoxic cells gave large reductions in TCP. (4) Restricted dose redistribution still gave a substantial increase in TCP as compared to uniform dose boosts. In conclusion, redistributing dose according to tumour oxygenation status might increase TCP when the tumour response to radiotherapy is limited by chronic hypoxia. This could potentially improve treatment outcome in a subpopulation of patients who respond poorly to conventional radiotherapy.

Cell Hypoxia↗
PubMed2004

Effectiveness of two-liquid silane primers on bonding sintered feldspathic porcelain with a dual-cured composite luting agent.

The purpose of the current study was to evaluate the effect of two-component ceramic primers on the bond strength of a composite material joined to a sintered porcelain material. Two sizes of porcelain discs (VMK 68; 10 and 8 mm in diameter by 2.5 mm thick) were ground (No. 1000 SiC), and surface-conditioned using one of the following systems: (i) unprimed, (ii) Liquid A of the Porcelain Liner M material (PLM-A), (iii) Liquid B of Porcelain Liner M (PLM-B), (iv) Liquids A and B of Porcelain Liner M (PLM-AB), (v) Liquid A of the Tokuso Ceramics Primer material (TCP-A), (vi) Liquid B of Tokuso Ceramics Primer (TCP-B) and (vii) Liquids A and B of Tokuso Ceramics Primer (TCP-AB). All specimens were bonded with the Bistite II dual-cured composite luting agent, and shear bond strengths were determined both after 24 h immersion in water and after subsequent thermocycling (4-60 degrees C, 1 min each, 20,000 cycles). Of the seven groups assessed, two groups (PLM-AB and TCP-AB) showed the greatest bond strength both before (30.7 MPa for PLM-AB and 29.7 MPa for TCP-AB) and after (19.7 MPa for PLM-AB and 22.4 MPa for TCP-AB) thermocycling (P < 0.05). No significant differences were found between the PLM-AB and TCP-AB groups regardless of the application of thermocycling (P > 0.05). It can be concluded that both of the acid-catalysed two-liquid ceramic primers showed superior bonding to the unhydrolysed single-liquid silane agents (PLM-B and TCP-A) when the feldspathic porcelain was bonded with the Bistite II luting composite.

Analysis of Variance↗
PubMed1991

Glycine-like modulation of N-methyl-D-aspartate receptors by a monoclonal antibody that enhances long-term potentiation.

We have identified a monoclonal antibody, B6B21, that significantly elevates long-term potentiation when applied to CA1 pyramidal cell apical dendrites in rat hippocampal slices and characterized its binding to N-methyl-D-aspartate-receptor complexes using extensively washed hippocampal membranes. Five micrograms of affinity-purified B6B21 per 100 micrograms of membranes gave a two- to threefold elevation in N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) binding. When [3H]TCP binding was stimulated by the combined addition of maximal concentrations of glutamate, glycine, and magnesium, B6B21 no longer stimulated [3H]TCP binding. Like glycine, B6B21 enhanced the effect of N-methyl-D-aspartate and glutamate in stimulating [3H]TCP binding. Moreover, B6B21 reversed 7-chlorokynurenic acid inhibition of [3H]TCP binding, but it had no effect on the inhibition of [3H]TCP binding by D-(-)-2-amino-5-phosphonovaleric acid. B6B21 increased the rate of association and dissociation of [3H]TCP, but had no effect on equilibrium binding. Glutamate, but not glycine, however, increased B6B21-enhancement of [3H]TCP association and dissociation. B6B21 binding at strychnine-insensitive glycine sites was confirmed by direct measurement of [3H]glycine binding. These results suggest that B6B21 binds directly to N-methyl-D-aspartate receptors and displays properties similar to glycine.

Animals↗
PubMed1991

A comparison of [3H]MK-801 and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine binding to the N-methyl-D-aspartate receptor complex in human brain.

The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed2004

Effect of a test and control program for bovine Johne's disease in Victorian dairy herds 1992 - 2002.

OBJECTIVE: To report on progress in Johne's disease (JD) control in infected dairy herds participating in the Victorian Johne's disease Test and Control Program (TCP). PROCEDURE: Clinical histories and JD testing data recorded by the Department of Natural Resources and Environment (now called Department of Primary Industries) were analysed for 542 dairy herds participating in the TCP. The herds were required to conduct annual herd tests of cattle 2 years old and older with an enzyme linked immunosorbent assay (ELISA), cull the reactors and manage the younger cattle to minimise infection. RESULTS: Testing of over 680,000 animals identified over 10,000 reactors giving an average prevalence of reactors at the first whole-herd test (T1) of 1.78%. There was a relatively rapid increase in the incidence of clinical disease before the TCP started and then it markedly declined. There was a slow and interrupted decline in reactor prevalence, with a marked peak occurring at the fourth herd test (T4). The average age of reactors and clinical cases was 5.7 and 5.9 years, respectively. Of the reactors and clinical cases detected during the TCP, 87% and 95% respectively, were born before the TCP started. Thirty herds completed the program by achieving three successive negative whole herd tests and 91 herds dropped out because of inability to comply with the agreed requirements of the program. There were no home-bred reactors born after the start of the program in 253 (47%) herds and of the 522 herds that were tested more than once, there were 319 (61%) herds in which no home-bred reactors were detected after the first year of testing. The number of ELISA positive animals detected at T1 appeared to be only about 26% of the animals from that round that subsequently became positive or developed clinical disease at later test rounds. CONCLUSION: The TCP caused a marked decline in the number of clinical cases, probably because animals in which clinical disease was imminent were detected by testing and removed. A reduction in prevalence of reactors occurred only when most herd members were born after the TCP started. The sensitivity of the ELISA appears to be low based on the large number of reactors that were negative at T1 but were positive at later tests. Low sensitivity of diagnostic tests and the long incubation period of the disease limits meaningful analysis of the program until it has continued for some years. Measures adopted in the TCP have not broken the cycle of infection in many participating herds. It is unsure if this was because of poor compliance with control recommendations or a poor understanding of methods of transmission by scientists. Eradication is not feasible in the short-term.

Animals↗
PubMed2002

On cold spots in tumor subvolumes.

Losses in tumor control are estimated for cold spots of various "sizes" and degrees of "cold dose." This question is important in the context of intensity modulated radiotherapy where differential dose-volume histograms (DVHs) for targets that abut a critical structure often exhibit a cold dose tail. This can be detrimental to tumor control probability (TCP) for fractions of cold volumes even as small as 1%, if the cold dose is lower than the prescribed dose by substantially more than 10%. The Niemierko-Goitein linear-quadratic algorithm with gamma50 slope 1-3 was used to study the effect of cold spots of various degrees (dose deficit below the prescription dose) and size (fractional volume of the cold dose). A two-bin model DVH has been constructed in which the cold dose bin is allowed to vary from a dose deficit of 1%-50% below prescription dose and to have volumes varying from 1% to 90%. In order to study and quantify the effect of a small volume of cold dose on TCP and effective uniform dose (EUD), a four-bin DVH model has been constructed in which the lowest dose bin, which has a fractional volume of 1%, is allowed to vary from 10% to 45% dose deficit below prescription dose. The highest dose bin represents a simultaneous boost. For fixed size of the cold spot the calculated values of TCP decreased rapidly with increasing degrees of cold dose for any size of the cold spot, even as small as 1% fractional volume. For the four-subvolume model, in which the highest dose bin has a fractional volume of 80% and is set at a boost dose of 10% above prescription dose, it is found that the loss in TCP and EUD is moderate as long as the cold 1% subvolume has a deficit less than approximately 20%. However, as the dose deficit in the 1% subvolume bin increases further it drives TCP and EUD rapidly down and can lead to a serious loss in TCP and EUD. Since a dose deficit to a 1% volume of the target that is larger than 20% of the prescription dose may lead to serious loss of TCP, even if 80% of the target receives a 10% boost, particular attention has to be paid to small-volume cold regions in the target. The effect of cold regions on TCP can be minimized if the EUD associated with the target DVH is constrained to be equal to or larger than the prescription dose.

Algorithms↗
PubMed1998

Analysis of clinical and environmental strains of nontoxigenic Vibrio cholerae for susceptibility to CTXPhi: molecular basis for origination of new strains with epidemic potential.

Toxigenic Vibrio cholerae strains are lysogens of CTXPhi, a filamentous phage which encodes cholera toxin. The receptor for CTXPhi for invading V. cholerae cells is the toxin-coregulated pilus (TCP), the genes for which reside in a larger genetic element, the TCP pathogenicity island. We analyzed 146 CTX-negative strains of V. cholerae O1 or non-O1 isolated from patients or surface waters in five different countries for the presence of the TCP pathogenicity island, the regulatory gene toxR, and the CTXPhi attachment sequence attRS, as well as for susceptibility of the strains to CTXPhi, to investigate the molecular basis for the emergence of new clones of toxigenic V. cholerae. DNA probe or PCR assays for tcpA, tcpI, acfB, toxR, and attRS revealed that 6.85% of the strains, all of which belonged to the O1 serogroup, carried the TCP pathogenicity island, toxR, and multiple copies of attRS, whereas the remaining 93.15% of the strains were negative for TCP but positive for either one or both or neither of toxR and attRS. An analysis of the strains for susceptibility to CTXPhi, using a genetically marked derivative of the phage CTX-KmPhi, showed that all TCP-positive CTX-negative strains and 1 of 136 TCP-negative strains were infected by the phage either in vitro or in the intestines of infant mice. The phage genome integrated into the chromosome of infected V. cholerae O1 cells forming stable lysogens. Comparative analysis of rRNA gene restriction patterns revealed that the lysogens derived from nontoxigenic progenitors were either closely related to or distinctly different from previously described clones of toxigenic V. cholerae. To our knowledge, this is the first demonstration of lysogenic conversion of naturally occurring nontoxigenic V. cholerae strains by CTXPhi. The results of this study further indicated that strains belonging to the O1 serogroup of V. cholerae are more likely to possess the TCP pathogenicity island and hence to be infected by CTXPhi, leading to the origination of potential new epidemic clones.

Animals↗
PubMed2005

Identification of a TcpC-TcpQ outer membrane complex involved in the biogenesis of the toxin-coregulated pilus of Vibrio cholerae.

The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis proteins revealed that TcpC was absent specifically in a tcpQ mutant. TcpQ is a predicted periplasmic protein required for TCP biogenesis. Fractionation studies revealed that the protein is not localized to the periplasm but is associated predominantly with the outer membrane fraction. An analysis of the amount of TcpQ present in the series of tcp mutants demonstrated the inverse of the TcpC result (absence of TcpQ in a tcpC deletion strain). Complementation of the tcpQ deletion restored TcpC levels and TCP formation, and similarly, complementation of tcpC restored TcpQ. Metal affinity pull-down experiments performed using His-tagged TcpC or TcpQ demonstrated a direct interaction between TcpC and TcpQ. In the presence of TcpQ, TcpC was found to form a high-molecular-weight complex that is stable in 2% sodium dodecyl sulfate and at temperatures below 65 degrees C, a characteristic of secretin complexes. Fractionation studies in which TcpC was overexpressed in the absence of TcpQ showed that TcpQ is also required for proper localization of TcpC to the outer membrane.

Amino Acid Sequence↗
PubMed2006

Association of cathepsin B gene polymorphisms with tropical calcific pancreatitis.

BACKGROUND AND AIMS: Tropical calcific pancreatitis (TCP) is a type of chronic pancreatitis unique to countries in the tropics. Mutations in pancreatic secretory trypsin inhibitor (SPINK1) rather than cationic trypsinogen (PRSS1) explain the disease in only 50% of TCP patients. As cathepsin B (CTSB) is known to activate cationic trypsinogen, we attempted to understand the role of CTSB mutations in TCP. Evidence of epistatic interaction was investigated with the previously associated N34S SPINK1 allele, a variant considered to be a modifier rather than a true susceptibility allele. SUBJECTS AND METHODS: We sequenced the coding region of CTSB gene in 51 TCP patients and 25 controls and further genotyped 89 patients and 130 controls from the same cohort for Leu26Val, C595T, T663C, and Ser53Gly polymorphisms. The positive findings observed in the earlier cohort were re-examined in an ethnically matched replication cohort comprising 166 patients and 175 controls. Appropriate statistical analyses were performed and Bonferroni correction for multiple testing was applied. RESULTS: We found a statistically significant association of the Val26 allele at Leu26Val polymorphism with an odds ratio (OR) of 2.15 (95% confidence interval (CI) 1.60-2.90 (p = 0.009)), after Bonferroni correction (corrected p value = 0.025). This significant association of Leu26Val with TCP was replicated in another cohort (OR 2.10 (95% CI 1.56-2.84); p = 0.013). Val26 allele also showed significantly higher frequency in N34S positive and N34S negative patients than in controls (p = 0.019 and 0.013, respectively). We also found significant differences in the mutant allele frequencies at Ser53Gly and C595T single nucleotide polymorphisms between N34S positive patients and controls (p = 0.008 and 0.001, respectively). Although haplotype analysis did not complement the results of allelic association, it did uncover a unique haplotype protective for TCP (p = 0.0035). CONCLUSION: Our study suggests for the first time that CTSB polymorphisms are associated with TCP. As PRSS1 mutations are absent in TCP and the N34S SPINK1 mutation is proposed to play a modifier role, these variants may be critical as a trigger for cationic trypsinogen activation.

Calcinosis↗
PubMed1990

Effects of hypoxia-ischemia and MK-801 treatment on the binding of a phencyclidine analogue in the developing rat brain.

The phencyclidine analogue [3H](1-[2-thienyl]cyclohexyl)piperidine (3H-TCP) binds to the ion channel associated with the N-methyl-D-aspartate receptor channel complex. In vitro autoradiography indicates that the distribution of 3H-TCP binding in brain closely parallels that of [3H]glutamate binding to the N-methyl-D-aspartate receptor. In nine 7-day-old rats, an acute focal hypoxic-ischemic insult produced by unilateral carotid artery ligation and subsequent exposure to 8% oxygen acutely reduced 3H-TCP binding ipsilateral to the ligation by 30% in the CA1, by 27% in the CA3, by 26% in the dentate gyrus, and by 17% in the striatum compared with values from the contralateral hemisphere. In 10 littermates that received 1 mg/kg of the neuroprotective noncompetitive N-methyl-D-aspartate antagonist MK-801 immediately before hypoxic exposure, the regional distribution of 3H-TCP binding in hypoxic-ischemic brain was relatively preserved and there were no interhemispheric asymmetries in 3H-TCP binding densities. In addition, in three unoperated rats decapitated 24 hours after MK-801 treatment, 3H-TCP binding was reduced by 15-35%; similar bilateral suppression of 3H-TCP binding was detected in MK-801-treated ligates. Our data indicate that 3H-TCP autoradiography can be used to assay the efficacy of neuroprotective agents in this experimental model of perinatal stroke.

Animals↗
PubMed1994

Alpha-tocopherol inhibits agonist-induced monocytic cell adhesion to cultured human endothelial cells.

Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: IL-1 (10 ng/ml), LPS (10 ng/ml), thrombin (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an IL-1-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of E-selectin which is consistent with the ability of a monoclonal antibody to E-selectin to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. Protein kinase C (PKC) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of PKC substrates was observed. Activation of the transcription factor NF-kappa B is reported to be necessary but not sufficient for E-selectin expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp-induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp.

Base Sequence↗
PubMed1989

[Metabolism and urinary excretion of chlorpyrifos in rats].

Urinary metabolites and biological half-life of chlorpyrifos (O, O-diethyl-O-3,5,6-trichloro-2-pyridinyl phosphorothioate) were investigated. Male Wistar rats weighing 200 g were intraperitoneally injected with chlorpyrifos at a level of 0.2 mmol/kg body weight. Both chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) levels in blood showed maximum values at 5 h post-injection, and then decreased rapidly. Biological half-lives of the blood chlorpyrifos and TCP were estimated to 8.15 and 24.66 h, respectively. Urine was collected for 96 h post-injection and hydrolyzed with 4 N HCl or beta-glucuronidase with sulfatase, and TCP released was determined. Urinary excretion levels of the acid hydrolysis-released TCP and the enzyme hydrolysis-released TCP accounted for 86 and 54% of chlorpyrifos administered, respectively. Urinary excretion levels of alkylphosphate for 96 h post-injection were analyzed. The excretion levels of diethylthiophosphate (DETP) and diethylphosphate (DEP) accounted for 45 and 15% of chlorpyrifos administered, respectively. These results indicate that 1) about half of the chlorpyrifos administered was directly hydrolyzed to DETP and TCP, 2) 10 to 20% was hydrolyzed to DEP and TCP after the oxidation to chlorpyrifos oxon, and 3) about 30% was dealkylated to TCP-phosphate after the oxidation.

Animals↗