Gαq/p63RhoGEF interaction in RhoA/Rho kinase signaling: investigation in Gitelman's syndrome and implications with hypertension.

PURPOSE: Gitelman's syndrome (GS) presents normo-hypotension and absence of cardiovascular-renal remodeling despite high angiotensin II (Ang II), activation of renin-angiotensin-aldosterone system and is a human model of endogenous antagonism of Ang II signaling, opposite to hypertension. GS's clinical presentation leads to questions regarding what features might be responsible. One area of investigation involves Ang II signaling. In hypertensive patients, RhoA/Rho kinase (RhoA/ROCK) pathway activation by Ang II is involved in hypertension development/maintenance and induction of long-term consequences (cardiovascular-renal remodeling), while GS has reduced p63RhoGEF gene and protein levels and ROCK activity. Ang II signaling is mediated by Gαq, which interacts with p63RhoGEF via the α6-αN linker connecting p63RhoGEF's DH and PH domains acting as a conformational switch to activate RhoA/ROCK signaling. METHODS: We have investigated in GS patients, the presence of mutations in either p63RhoGEF's α6-αN linker domain and in Gαq's Ala253, Trp263, and Tyr356 residues, crucial for p63RhoGEF-Gαq interplay. RESULTS: No mutations have been found in specific aminoacids of p63RhoGEF α6-αN linker and Gαq, key for p63RhoGEF/Gαq interplay. CONCLUSIONS: Gitelman's syndrome normo/hypotension and lack of cardiovascular-renal remodeling are not due to mutations of p63RhoGEF α6-αN linker and Gαq interactions. This opens the way for investigations on different coding and no-coding regions (p63RhoGEF and Gαq promoters) and on altered transcriptional/post-transcriptional regulation. Clarification of how these biochemical/molecular mechanisms work/interact would provide insights into mechanisms involved in the GS's Ang II signaling fine tuning, in human physiology/pathophysiology in general and could also identify significant targets for intervention in the treatments of hypertension.

The temporal relations across burnout dimensions in athletes.

Burnout is a construct that has garnered considerable attention in sport psychology within recent years. Several hypothesized models regarding how the three dimensions (exhaustion, devaluation, and reduced sense of accomplishment) temporally relate to each other have been advanced. One proposal outlined by Maslach and Leiter suggests that exhaustion predicts devaluation which predicts reduced sense of accomplishment. However, there is no consensus among researchers as it has been argued that exhaustion predicts devaluation and reduced accomplishment separately. The aim of this study was to test multiple alternative hypotheses regarding the relationships of the burnout dimensions in athletes. Two samples of Swedish youth elite athletes with differing time spans between measurements were used. Specifically, one sample involved time-intensive measures collected every week over an eight-week period, and the other sample included four measurement points across an 18-month period. Results showed that none of the previously proposed models outlining the temporal relations of burnout dimensions were supported. Statistical analysis of the models including the cross-lagged predictions of dimensions did not have any statistically significant impact except when exhaustion negatively predicted devaluation between time 1 (month 0) and time 2 (month 6) in the 18-month sample; this relation faded in the following time points. Further, issues regarding the stability of devaluation and reduced sense of accomplishment emerged as their autocorrelation were very weak in the time-intensive sample. These findings raise a number of points for further theoretical and practical discussions about the athlete burnout construct.

Prognostic value of hypoxia inducible factor 1α in esophageal squamous cell carcinoma.

Hypoxia inducible factor 1α (HIF 1α) plays a major role in the pleitropic response observed secondary to hypoxic conditions in tumors. Its expression in the tumor cells has been correlated to tumor aggressiveness and prognosis in squamous cell carcinoma (SCC) of the esophagus in Far Eastern population, but limited information is available on the prognostic role of HIF 1α in SCC of esophagus in European population. This information may help in choosing appropriate therapeutic strategies and possibly developing a monoclonal antibody with therapeutic potential targeting the HIF 1α. Tumor samples from 36 patients diagnosed with SCC of the esophagus were collected. Prepared tissue sections were stained with validated and specific monoclonal antibodies for HIF 1α and the expression was correlated with the disease pattern and survival. Out of 36 patients, 17 patients showed low and 19 high expression of HIF 1α. There was no difference in the disease-free and overall survival between these two groups (P > 0.05, log rank test). Regression analysis showed that HIF 1α was not an independent prognostic factor for survival (P > 0.05). HIF 1α did not show prognostic value in SCC of the esophagus in our study on European population, in agreement with previous studies. Novel strategies on the therapeutic manipulation of HIF 1α in cancer are to be explored further and may have a role to play in improving treatment outcome.

Isolation of Salmonella spp. from Animal Feed.

The isolation of Salmonella from feed is challenging and adjustments need to be made in order to accurately isolate the pathogen from feed. This is due to the complex nature of the feed matrix, which is both porous and fibrous. The outlined method below contains the essential components of a successful Salmonella methodology for the analysis of feed that overcomes the limitations of currently available methods.

Oxidative stress-related proteins in a Conn's adenoma tissue. Relevance for aldosterone's prooxidative and proinflammatory activity.

BACKGROUND AND AIM: Angiotensin II (Ang II) induces oxidative stress (OxSt), which is essential for cardiovascular remodeling. Aldosterone also induces fibrosis and remodeling through direct effect on non-classical mineralocorticoid (MR) target tissues. However, studies on the role of aldosterone on OxSt and related factors in humans are lacking. MATERIALS AND METHODS: We assessed gene and protein expression of p22phox (RT-PCR and Western blot), NAD(P)H oxidase subunit essential for superoxide production and gene expression of transforming growth fator (TGF) beta, plasminogen activator inhibitor (PAI)-1, and heme oxygenase (HO)-1, effectors of OxSt (RT-PCR), in a Conn's adenoma, removed from a patient with primary hyperaldosteronism. Ang II type 1 (AT1R) and MR receptors expression were also evaluated (RT-PCR). The normal adrenal tissue adjacent to the adenoma was used as control. RESULTS: p22phox gene and protein expression were higher (31% and 53%, respectively) in the adrenal adenoma. TGFbeta, PAI-1, and HO-1 gene expression were also higher (25%, 129%, and 25%, respectively) in the adrenal adenoma while AT1R gene expression was similar (8%). The expression of MR in the adenoma was documented. CONCLUSIONS: This report demonstrates in a human model that the increased aldosterone production has effects on enzyme systems related to OxSt, enhancing the systemic fibrogenic effects of aldosterone excess through TGFbeta and PAI-1 expression which was previously demonstrated only indirectly in vitro and in animal models. The presence of MR expression in the adenoma may link the hormone with the adenoma growth. Therefore, the results of this study derived from a single case might represent an important working hypothesis for further research in a larger number of cases to clarify the role of aldosterone overproduction on OxSt and its clinical relevance.

Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells.

Exposure to high levels of ozone (O(3)) damages respiratory tract epithelial cells. This research evaluated the ability of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide free radical, to decrease O(3)-mediated injury to a respiratory tract-derived cell line (A549 cells) by monitoring in this cell system the interleukine-8 (IL-8) production. TEMPO reduced O(3)-induced IL-8 production in A549 cells, as evidenced by PCR analysis, Western blot and ELISA assays. This behaviour is explainable on the basis of the reactivity between TEMPO with O(3) and/or O(3)-derived free radicals in biological systems. The study provides evidence that TEMPO reacts with O(3) and/or its cytotoxic products and may provide protections against O(3)-induced biotoxicities.

High angiotensin II state without cardiac remodeling (Bartter's and Gitelman's syndromes): are angiotensin II type 2 receptors involved?

BACKGROUND/AIMS: While Angiotensin II (Ang II) is a major factor in the development of cardiomyocyte hypertrophy and a pivotal role for Ang II signals via ERK1/2 has been identified, mechanism(s) responsible are still unclear. As Bartter's and Gitelman's syndrome patients (BS/GS) have increased Ang II, and yet normo/hypotension, hyporesponsiveness to pressors and blunted Ang II signaling via type 1 receptors (AT1R), this study assesses BS/GS's left ventricular (LV) mass and structure as well as Ang II induced ERK1/2 phosphorylation compared with essential hypertensive patients (EH) and normotensive healthy subjects (C) to gain insight into Ang II mediated processes. METHODS: Indices of cardiac hypertrophy were determined by M-mode, two-dimensional echo Doppler and ERK phosphorylation by Western blot. RESULTS: None of BS/GS exhibited LV remodelling; LV mass, LV end-diastolic volume and mass/volume ratio were unchanged vs C (60+/-14 g/m2 vs 64+/-12, 64+/-12 ml/m2 vs 60+/-8 and 0.95+/-0.2 vs 1.0+/-0.2, respectively) and reduced vs EH (119+/-15, p<0.001, 78+/-9, p<0.05 and 1.52+/-0.15, p<0.01). Despite BS/GS's higher plasma renin activity and aldosterone and unchanged level of AT1R, Ang II induced ERK1/2 phosphorylation was reduced vs both C and EH: 0.64 d.u.+/-0.08 vs 0.90+/-0.06 in C, p<0.006, and vs 1.45+/-0.07 in EH, p<0.001. CONCLUSION: The data point to a direct cardioremodeling role for Ang II and support a role of Ang II type 2 receptor (AT2R) signaling as involved in the lack of cardiovascular remodeling in BS/GS. However, further studies using more direct approaches to demonstrate the effects of AT2R signaling must be pursued.

International study on the validation of models for the environmental transfer of tritium and carbon-14.

The International Atomic Energy Agency (IAEA)'s Environmental Modelling for Radiation Safety (EMRAS) model evaluation programme includes a Tritium and Carbon-14 Working Group (TCWG), the goal of which is to test the performance of models for the environmental transfer. This paper describes work on two of the nine TCWG scenarios; one involved the prediction of time-dependent tritium concentrations in freshwater mussels subject to an abrupt change in ambient tritium levels and the second was concerned with the prediction of carbon-14 concentrations in rice grown in the vicinity of sources of continuous atmospheric releases.

Carbon-14 transfer into rice plants from a continuous atmospheric source: observations and model predictions.

Carbon-14 (14C) is one of the most important radionuclides from the perspective of dose estimation due to the nuclear fuel cycle. Ten years of monitoring data on 14C in airborne emissions, in atmospheric CO2 and in rice grain collected around the Tokai reprocessing plant (TRP) showed an insignificant radiological effect of the TRP-derived 14C on the public, but suggested a minor contribution of the TRP-derived 14C to atmospheric 14C concentrations, and an influence on 14C concentrations in rice grain at harvest. This paper also summarizes a modelling exercise (the so-called rice scenario of the IAEA's EMRAS program) in which 14C concentrations in air and rice predicted with various models using information on 14C discharge rates, meteorological conditions and so on were compared with observed concentrations. The modelling results showed that simple Gaussian plume models with different assumptions predict monthly averaged 14C concentrations in air well, even for near-field receptors, and also that specific activity and dynamic models were equally good for the prediction of inter-annual changes in 14C concentrations in rice grain. The scenario, however, offered little opportunity for comparing the predictive capabilities of these two types of models because the scenario involved a near-chronic release to the atmosphere. A scenario based on an episodic release and short-term, time-dependent observations is needed to establish the overall confidence in the predictions of environmental 14C models.

Clinical investigation of a cytostatic calcium influx inhibitor in patients with refractory cancers.

Carboxyamido-triazole (CAI) is a synthetic inhibitor of non-excitable calcium channels that reversibly inhibits angiogenesis, tumor cell proliferation, and metastatic potential. Inhibition of calcium influx and calcium-dependent events is a potential common mechanism underlying these effects of CAI. The cytostatic and antiangiogenic properties of CAI led to its development for clinical investigation. In a Phase I clinical trial open to patients with refractory solid tumors, 49 patients received p.o. administered CAI daily or every other day. Two oral formulations, PEG-400 CAI solution and a gelatin capsule containing CAI in PEG-400, were tested. All administered dosages of CAI yielded plasma concentration at or above the range demonstrated to be effective in inhibiting signaling and cancer progression in vitro and in preclinical models (1 microgram/ml, 2.3 microM). Toxicity of p.o. administered CAI most commonly consisted of dose-related grade 1-2 nausea, vomiting, and occasional anorexia. CAI administration at bedtime ameliorated gastrointestinal complaints in many patients; others required addition of simple antiemetic regimens, usually consisting of metoclopropamide or prochlorperazine. Gastrointestinal complaints were the cause for compliance-limiting toxicity at 175 mg/m2/day of the liquid formulation and 125 mg/m2/day of the gelatin capsule formation. Reversible and rare sensory axonal neuropathy (grade 3, 1 patient) and neutropenia (grade 4, 1 patient) were dose-limiting toxicities observed at the 330 mg/m2 every-other-day liquid CAI dose level. No evidence of cumulative end organ damage or central nervous system injury was observed. Disease stabilization and improvement in performance status was observed. Disease stabilization and improvement in performance status was observed in 49% of evaluable patients who had disease progression before CAI. Disease stabilization and associated improvement in performance status was seen in patients with renal cell carcinoma (7 months), pancreaticobiliary carcinomas (3, 5, and 5 months), melanoma (7 months), ovarian cancer (7 months), and non-small cell lung cancer (3 months). The recommended Phase II doses from this trial are 150 mg/m2/day in the liquid formation and 100 mg/m2/day in the gelatin capsule formation.

Estimating cut points: A simple method for new wearables.

UNLABELLED: Wearable technology is readily available for continuous assessment due to a growing number of commercial devices with increased data capture capabilities. However, many commercial devices fail to support suitable parameters (cut points) derived from the literature to help quantify physical activity (PA) due to differences in manufacturing. A simple metric to estimate cut points for new wearables is needed to aid data analysis. OBJECTIVE: The purpose of this pilot study was to investigate a simple methodology to determine cut points based on ratios between sedentary behaviour (SB) and PA intensities for a new wrist worn device (PRO-Diary™) by comparing its output to a validated and well characterised 'gold standard' (ActiGraph™). STUDY DESIGN: Twelve participants completed a semi-structured (four-phase) treadmill protocol encompassing SB and three PA intensity levels (light, moderate, vigorous). The outputs of the devices were compared accounting for relative intensity. RESULTS: Count ratios (6.31, 7.68, 4.63, 3.96) were calculated to successfully determine cut-points for the new wrist worn wearable technology during SB (0-426) as well as light (427-803), moderate (804-2085) and vigorous (≥ 2086) activities, respectively. CONCLUSION: Our findings should be utilised as a primary reference for investigations seeking to use new (wrist worn) wearable technology similar to that used here (i.e., PRO-Diary™) for the purposes of quantifying SB and PA intensities. The utility of count ratios may be useful in comparing devices or SB/PA values estimated across different studies. However, a more robust examination is required for different devices, attachment locations and on larger/diverse cohorts.

Transcription initiation by Escherichia coli RNA polymerase at the gene II promoter of M13 phage: stability of ternary complex, direct photocrosslinking to nascent RNA, and retention of sigma subunit.

The initial stages of transcription have been characterized using a template containing the gene II promoter region of M13 phage. Initiation of transcription in the presence of all four nucleotides gives rise to the 140-residue run-off transcript, with a minor pause at the RNA hexamer stage. Cycling, leading to the accumulation of significant amounts of short oligonucleotides [1], was not observed. An RNA hexamer GUUUUU was the sole product when GpU and UTP were used and the ternary complex with the hexamer was stable and resistant to high salt (0.4 M) and S1 nuclease attack. After direct ultraviolet photocrosslinking of the RNA hexamer to RNA polymerase in the ternary complex, the radioactive label incorporation into various subunits was determined by autoradiography after sodium tetradecyl sulfate gel electrophoresis to be as follows: sigma, 86%; beta, 14%; beta' and alpha, negligible. Both electrophoresis and sucrose gradient centrifugation experiments indicate that the sigma subunit is not released from the ternary complex when either the RNA hexamer or the 140-residue RNA is synthesized on this template, even though the complexes are stable.

Apolipoprotein and size heterogeneity in human umbilical cord blood low density lipoproteins.

Neonatal umbilical cord blood plasma low density lipoproteins (LDL, d = 1.019-1.063 g/ml) were subfractionated by density gradient ultracentrifugation into seven fractions (from 1.024 to 1.062 g/ml); the bulk of the LDL mass was in a density region of 1.034-1.042 g/ml. Apolipoprotein B by 10% SDS-polyacrylamide gel electrophoresis varied inversely with density, with only trace amounts present in the most dense fraction. The distribution of apolipoprotein B molecular weight forms was assessed by both 3% SDS-polyacrylamide gel electrophoresis and relative aminoacyl mass determination. Lower molecular weight forms of apolipoprotein B (B74 and B26) increased relative to apolipoprotein B100 with increasing density, ranging from undetectable in fraction 1 to apolipoproteins B26 and B74 comprising 30% of the total mass of apolipoprotein B in fraction 6. No apolipoprotein B48 was detectable in the LDL. Apolipoprotein E as determined by both SDS-polyacrylamide gel electrophoresis and radioimmunoassay increased with density with a maximum (14% of the protein) in the most dense fraction, fraction 7. Apolipoprotein A-I by SDS-polyacrylamide gel electrophoresis increased with increasing density and was the major apolipoprotein in fraction 7. Electron microscopic analysis revealed spherical particles whose diameters decreased with increasing density, ranging from 28.6 nm in the top fraction (fraction 1) to 15.6 nm in the bottom fraction (fraction 7). Gradient gel electrophoresis revealed that most of the fractions contained several different sized particles. The bottom fraction (fraction 7), enriched in apolipoproteins E and A-I, had a unique, poorly defined peak at 14.6 nm on gradient gel electrophoresis and showed a tendency to pack hexagonally upon electron microscopy. The unusual composition and apolipoprotein distribution in neonatal LDL fractions suggests that the LDL in the neonate are metabolically very diverse.

Phase I trial of micronized formulation carboxyamidotriazole in patients with refractory solid tumors: pharmacokinetics, clinical outcome, and comparison of formulations.

PURPOSE: Cytostatic agents targeted against angiogenesis and tumor cell invasive potential form a new class of investigational drugs. Orally administered carboxyamidotriazole (CAI) (NSC609974) is both antiangiogenic and antimetastatic. An encapsulated micronized powder formulation has been developed to optimize CAI administration. A phase I dose escalation trial with pharmacokinetic analysis has been performed. PATIENTS AND METHODS: Twenty-one patients with refractory solid tumors and good end organ function and performance status were enrolled onto the study. Patients received a test dose followed 1 week later by daily administration of CAI in the encapsulated micronized formulation at doses of 100 to 350 mg/m2. Patients remained on CAI until disease progression or dose-limiting toxicity. Plasma samples were taken to characterize the pharmacokinetic parameters of this formulation of CAI. RESULTS: All patients were assessable for toxicity and 18 were assessable for pharmacokinetics and response analysis. Grade 1 and 2 gastrointestinal side effects were observed in up to 50% of patients. Dose-limiting toxicity was observed in both patients treated at 350 mg/m2/d, consisting of reversible grade 2 to 3 cerebellar ataxia (n = 1) and confusion (n = 1). One minor response (MR) was observed in a patient with renal cell carcinoma and another nine patients had disease stabilization (MR + SD = 47%). Pharmacokinetic analysis demonstrated reduced bioavailability (58% reduction) compared with the PEG-400 liquid formulation previously reported. CONCLUSION: The better toxicity profile of encapsulated micronized CAI with similar frequency of disease stabilization and ease of administration compared with the liquid or gelatin capsule, suggests that the micronized formulation is a preferable formulation for subsequent studies. A dose of 300 mg/m2/d is proposed for phase II investigations.

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.

Bacteriophage T7 RNA polymerase and its active-site mutants. Kinetic, spectroscopic and calorimetric characterization.

It has been demonstrated that the amino acids Asp537, Asp812, Lys631, His811 and Tyr639 are involved in bacteriophage T7 RNA polymerase catalysis. In the present paper, we report kinetic, spectroscopic and calorimetric characterization of the wild-type and mutant T7 RNA polymerases generated at these five loci (D537N, E; K631M, R; Y639F, S, A, W; H811Q, A; D812N, E). The wild-type enzyme has a substantial amount of secondary structure as determined by CD analysis (alpha-helix, 43%; beta-sheet, 14%; beta-turn, 25%; unordered, 18%). The CD spectra of 12 mutants at five loci are very similar to that of the wild-type, except for the mutant Y639W. Within experimental error, the thermal transition temperatures measured by CD and DSC as well as the lambda max values of the fluorescence spectra were the same for the wild-type and all of the mutants. Therefore, the overall folding and stability of the mutant enzymes are very similar to those of the wild-type enzyme, although small local conformational changes cannot be excluded. For the synthesis of the pentamer pppGGACU, the mutants D537E and D812E showed an approximately two- to threefold decrease in (kcat)app and an approximately two- to threefold increase in (Km)app, relative to the wild-type, in contrast to the mutants D537N and D812N which exhibited no detectable activity. The mutant K631R showed a sevenfold reduction in (kcat)app and a two- to threefold increase in (Km)app, supporting our earlier observation with the mutant K631M that Lys631 may be involved in phosphodiester bond formation. The mutant Y639S can synthesize the trimer GGA with an approximately 50-fold decrease in (kcat)app and a tenfold increase in (Km)app, relative to the wild-type, underlining the importance of the phenyl ring of Tyr639. The mutant H811A, in which the side-chain at position 811 is incapable of forming a hydrogen bond, can synthesize the trimer GGA with an approximately tenfold decrease in (kcat)app and an approximately 35-fold increase in (Km)app. Thus, either the hydrogen-bonding capacity of this residue is non-essential or some other group can functionally substitute for the His811 side-chain. The wild-type enzyme showed significant effects of the base position in the sequence on the apparent binding constants for the NTPs. The kinetics of GpG-primed trimer, tetramer and pentamer synthesis on three 22 bp templates were investigated for the wild-type and mutant enzymes with measurable activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics of orally administered carboxyamido-triazole, an inhibitor of calcium-mediated signal transduction.

Carboxyamido-triazole (CAI), inhibits proliferation, invasion, and metastatic potential of a number of cancer cell lines at concentrations greater than 0.4 microgram/ml. The objective of this study was to characterize the pharmacokinetic profile from the first Phase I clinical trial of CAI for the single test dose and multiple daily dosing schedule. Two different p.o. formulations (liquid and gelcap) of CAI were administered. Thirty-nine patients with cancer were enrolled. The dose escalation schema was 100, 125, and 150 mg/m2/day and 200 and 330 mg/m2 every other day of the liquid formulation, plus 100 and 125 mg/m2/day and 200 mg/m2 every other day of the gelcap. The CAI pharmacokinetics are best described by a two-compartment open linear model. The gelcap was more rapidly absorbed than the liquid [time to maximum plasma concentration (Tmax) = 2.06 +/- 1.02 versus 5.31 +/- 3.59 h, P2 = 0.0012] which resulted in higher peak plasma concentrations. There was no evidence of saturable elimination as the dose was increased. The mean steady-state peak concentration was 5.1 +/- 1.0 microgram/ml for the 150 mg/m2/day multiple daily dosing regimen. The terminal half-life of CAI was relatively prolonged, 111 h, and the total body p.o. clearance was low (1.87 liters/h). The peak concentration for all dose levels explored was greater than the targeted concentration suggested by in vitro data for activity. Thus, these data suggest that an effective cytostatic exposure of CAI may be obtained with daily or every other day dosing without severe toxicity.

The difference in gastric cancer between Japan, USA and Europe: what are the facts? what are the suggestions?

In Japan the survival rate for gastric cancer has steadily improved over the last 30 years whilst that in the West has remained static and inferior. In this review three hypotheses are examined to explain the difference. There is little evidence to suggest genetic differences, which might result in a less aggressive cancer in Japan. Recently there has been a rise in the proportion of cancers of the gastro-oesophageal junction in the West and this has not been seen in Japan. The comparison of survival data from these two regions is problematic with different staging systems and a stage migration effect. The established surgical treatment of gastric cancer in Japan is radical gastrectomy and regional lymphadenectomy and this has been proposed as a superior treatment to the standard gastrectomy common in the West. The results for survival benefit however, have not been reproduced in randomized clinical trials. The heterogeneity of adjuvant and neoadjuvant treatment regimens in Japan and the West has led to difficulties in the interpretation of their effects. There is considerable scope for future collaboration between clinicians in the West and Japan.

Redox regulation of wound healing? NF-kappaB activation in cultured human keratinocytes upon wounding and the effect of low energy HeNe irradiation.

The complex process of wound healing as well as the signaling systems orchestrating this intricate process remain incompletely defined. Using human keratinocytes in primary culture, we sought to characterize their NF-kappaB responses to wounding alone or in combination with other treatments. We initially characterized these cultured human keratinocytes responses to known NF-kappaB activators (PMA, TNF-alpha and IL-1) using two different assays, immunohistochemistry and electrophoretic mobility shift (EMSA). After eliciting the expected NF-kappaB responses, we applied these same assays to assess responses to either wounding or HeNe irradiation alone. The results obtained indicated that only a modest/sporadic activation of NF-kappaB was elicited by these which was only detectable using immunohistochemistry. When the combination of wounding and HeNe irradiation on NF-kappaB status was assessed, a marked, localized activation of NF-kappaB in keratinocytes along the wound edge was found. Treatment induced NF-kappaB activation (e.g., wounding, HeNe irradiation and combined wounding and HeNe irradiation) was abrogated by pyrrolidine dithiocarbamate (PDTC) which inhibits NF-kappaB activation through an as yet incompletely understood (antioxidant?) mechanism. These data therefore suggest that NF-kappaB and oxidation mediated changes in its activation state likely play important roles in normal cutaneous wound healing.