The impact of potential leakage from the sub-seabed CO2 storage site on the phosphorus transformation in marine sediments - An experimental study.

Carbon Capture and Storage (CCS) in the sub-seabed geological formations is a method of mitigation of carbon dioxide (CO2) emissions to avoid anthropogenic climate change. While CCS can be one of the most promising technologies to reduce atmospheric CO2 in the short and medium term, it raises serious concerns about the potential leakage of gas from storage sites. In the present study, the impact of acidification induced by CO2 leakage from a sub-seabed storage site on geochemical pools, and thus the mobility, of phosphorus (P) in sediment was investigated during laboratory experiments. The experiments were conducted in a hyperbaric chamber at a hydrostatic pressure of 900 kPa, which simulates pressure conditions at a potential sub-seabed CO2 storage site in the southern Baltic Sea. We performed three separate experiments in which the partial pressure of CO2 was: 352 µatm (corresponding pH = 7.7); 1815 µatm (corresponding pH = 7.0), and 9150 µatm (corresponding pH = 6.3). Under pH 7.0 and 6.3, apatite P is transformed into organic and non-apatite inorganic forms, which are less stable than CaP bonds and can be more easily released into the water column. At pH 7.7, P released during mineralization of organic matter and microbial reduction of FeP phases is bound with Ca, and the concentration of this form increases. The obtained results indicate that acidification of bottom water can reduce the efficiency of P burial in marine sediments, which contributes to an increase in P concentration in the water column and promote eutrophication especially in shallow areas.

The biological markers and results of treatment in male breast cancer patients. The Cracow experience.

Male breast cancer is a rare form of carcinoma with an incidence rate of approximately 0.5-1% compared with cases of breast carcinoma as a whole. Male breast cancer reacts effectively to endocrine therapy because of a high frequency of hormone receptor expression.The aim of the present study was the assessment of correlations between stage, grade, expression of steroid receptors, basal/mesenchymal markers and proliferation index, as well as analysis of the impact of the above-mentioned parameters on overall (OS) and disease-free survival (DFS) in the group of 32 male breast cancer patients, treated at the Centre of Oncology in Cracow.We showed the significant positive correlation between MIB-1 LI and tumor stage, and hormone receptors (ER or PgR) immunonegativity, and expression of EGFR, vimentin (p<0.05) and P-cadherin (the last at statistical border). The presence of any of basal or masenchymal markers correlated with a more advanced tumor stage. Moreover tumors without vimentin expression were characterised by lower MIB-1 LI and were more frequently EGFR immunonegative.We found that hormone receptor negativity, vimentin immunopositivity and high MIB-1 LI are significant independent indicators of poor OS and DFS for male breast cancer patients (p<0.05).

International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part I. Comparison with a traditional USP rotating disk apparatus.

A correlation of the logarithmic values of the in vitro dissolution rate, G, and the apparent solubility, S, was evaluated in phosphate and ammonium acetate buffer at an initial pH of 7. The dissolution rates were determined with a newly designed and build miniaturized rotating disk equipment, as well as with a traditional rotating disk apparatus. The two apparatuses gave the same correlation pattern of logG and logS. Thirteen diverse drug substances from all of the classes in the Biopharmaceutics Classification System (BCS) were used for the correlation in the phosphate buffer system, with the results from the miniaturized apparatus only. A coefficient of determination, R2, of 0.982 was found if bases formulated as hydrochloride salts were excluded in the correlation. The miniaturized equipment is used for rapid screening of the dissolution rate, approximately 10 min for one run, and consumes small amounts of substance (about 5 mg) and dissolution media. All quantifications were performed by using reversed phase high-performance liquid chromatography (RPHPLC) with a diode array detector (DAD), integrated with the miniaturized rotating disk equipment.

Correlation of in vitro dissolution rate and apparent solubility in buffered media using a miniaturized rotating disk equipment: Part II. Comparing different buffer media.

A correlation of the logarithmic values of the in vitro dissolution rate, G, and apparent solubility, S, was made for seven different drug substances from all of the classes in the Biopharmaceutics Classification System (BCS), in four different phosphate buffers. The effect of inorganic salts added as sodium chloride, sodium nitrate, sodium phosphate and sodium sulfate in the buffer media was investigated for the correlation. Triethanolammonium acetate buffer was also included in the study of the correlation of logG vs. logS. The pH was 7.0 ± 0.1 in all of the buffers to mimic a pH condition in intestinal fluids. The dissolution rate was determined with a newly constructed miniaturized rotating disk equipment, which enables fast determinations and consumes only minute quantities of substance (about 5 mg). The solubility was determined by conventional shake-flask methodology, using 1.5 mL solution volumes. All quantifications were performed with reversed phase high-performance liquid chromatography (RP-HPLC) and diode array detection (DAD). The different inorganic anions seemed to affect the solubility more than the dissolution rate. The phosphate and nitrate ions decreased the solubility for amines compared to the chloride ion. The best correlations of logG and logS were however obtained with a triethanolammonium acetate buffer. The good correlation (R2 = 0.991) may be sufficient in initial screening of drug solubility, based on dissolution rates in aqueous buffer media.

Seasonal variability of free amino acids in two marine bivalves, Macoma balthica and Mytilus spp., in relation to environmental and physiological factors.

The seasonal variability of the intracellular free amino acid (FAA) concentration was studied in 5 Macoma balthica populations and 7 Mytilus spp. populations along their European distribution. Because of the well known physiological role of FAA as organic osmolytes for salinity induced cell volume regulation in marine osmoconformers, FAA variations were compared in bivalve populations that were exposed to high vs. low intraannual salinity fluctuations. In general, seasonal FAA variations were more pronounced in M. balthica than in Mytilus spp. In both bivalve taxa from different locations in the Baltic Sea, highest FAA concentrations were found in autumn and winter and low FAA concentrations were measured in summer. Seasonal patterns were less pronounced in both taxa at locations with constant salinity conditions. In contrast to Baltic Sea populations, Atlantic and Mediterranean bivalves showed high FAA concentrations in summer and low values in winter, regardless of seasonal salinity fluctuations. Significant seasonal FAA variations at locations with constant salinity conditions showed that salinity appeared not to be the main factor in determining FAA concentrations. The seasonal patterns of the main FAA pool components, i.e. alanine, glycine and taurine, are discussed in the context of seasonal variations in environmental factors (salinity, temperature) and physiological state (glycogen content, reproductive stage).

Association of increased carotid intima-media thickness with the extent of coronary artery disease.

OBJECTIVE: To investigate (a) the relation between intima-media thickness (IMT) in carotid arteries and the extent of coronary artery disease (CAD); and (b) whether IMT is predictive of coronary atherosclerosis. The coexistence of severe extracranial atherosclerosis in patients with CAD was also analysed. METHODS: Coronary angiography and carotid ultrasound evaluation were performed in 558 consecutive patients (438 men), with a mean (SD) age of 58.8 (9.2) years and suspected CAD. Mean IMT was measured at both carotid arteries and expressed as the mean aggregate value. The relation between IMT and severity of CAD was determined. RESULTS: A significant correlation between mean IMT and advancing CAD (p < 0.0001) was found. Four independent predictors of CAD were found in the discriminant analysis: age (p = 0.0193), hyperlipidaemia (p < 0.0001), smoking (p = 0.0032), and IMT (p < 0.0001). A significant increase in IMT was observed among patients with one, two, and three vessel CAD. A log normal distribution of IMT values showed that if mean IMT was over 1.15 mm, patients had a 94% probability of having CAD, with sensitivity of 65% and specificity of 80% in the patients with a high risk of CAD. The number of critically stenosed extracranial arteries increased with advancing CAD. None of the patients with normal coronary arteries had severe stenosis of the extracranial arteries. Severe carotid, vertebral, or subclavian stenosis was found in 16.6% of patients with three vessel CAD. CONCLUSIONS: IMT increases with advancing CAD, patients with mean IMT over 1.15 mm have a 94% likelihood of having CAD, and the coexistence of CAD with severe stenosis of aortic arch arteries is relatively high and was found in 16.6% of patients with three vessel CAD.

Trace metals in the brown mussel Perna perna from the coastal waters off Yemen (Gulf of Aden): how concentrations are affected by weight, sex, and seasonal cycle.

The effects of seasonal cycle, sex of individuals, and changes of soft tissues weight on accumulated trace metal concentrations (Cd, Cu, Fe, Mn, Pb, Zn) were examined in the brown mussel Perna perna collected monthly from a natural rocky habitat in the coastal waters off Yemen, the Gulf of Aden, for a period of ten months. Basic hydrological parameters were recorded simultaneously. All metals analyzed displayed seasonal fluctuations with different temporal patterns and variable amplitudes. Similar seasonal cycles were observed for Cu, Mn, and Pb with an increase in accumulated concentration during the rainy period (NE monsoon), and a decrease thereafter. The concentrations of Cu, Mn, and partially Pb appeared to be related to environmental changes, the concentration of Pb possibly also being related to changes in body weight. Accumulated concentrations of Cu and Mn thus seem to reflect actual metal bioavailability in the ecosystem quite efficiently. The tissue levels of Fe and Cd changed inversely to fluctuations in body weight with additional variation due to monsoon-related environmental changes. The behaviors of Fe and Cd are therefore driven by seasonally changing body weight with a considerable contribution of external factors including fluctuations in hydrological conditions and metal exposure. The Zn concentrations tended to increase gradually throughout most of the year regardless of its concentration in the environment. Zinc is considered to be mainly regulated by physiological mechanisms in the mussel, making its accumulated metal concentration independent to some degree of environmental levels. Significant differences in trace metal concentrations between sexes (in favour of females) might have resulted from more intense formation of reproductive tissues and metal accumulation in sexual products of females during the prespawning and spawning periods.

Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia.

BACKGROUND: Mutations of the transforming growth factor beta (TGFbeta) receptor components ENDOGLIN and ALK-1 cause the autosomal dominant vascular disorder hereditary haemorrhagic telangiectasia (HHT). Heterozygous mutations of the type II receptor BMPR2 underlie familial primary pulmonary hypertension. OBJECTIVE: To investigate kindreds presenting with both pulmonary hypertension and HHT. METHODS: Probands and families were identified by specialist pulmonary hypertension centres in five countries. DNA sequence analysis of ALK-1, ENDOGLIN, and BMPR2 was undertaken. Cellular localisation was investigated by heterologous overexpression of mutant constructs in both BAEC and HeLa cells. The impact of a novel sequence variant was assessed through comparative analysis and computer modelling. RESULTS: Molecular analysis of 11 probands identified eight missense mutations of ALK-1, one of which was observed in two families. Mutations were located within exons 5 to 10 of the ALK-1 gene. The majority of ALK-1 mutant constructs appeared to be retained within the cell cytoplasm, in the endoplasmic reticulum. A novel GS domain mutation, when overexpressed, reached the cell surface but is predicted to disrupt conformational changes owing to loss of a critical hydrogen bond. Two novel missense mutations were identified in ENDOGLIN. CONCLUSIONS: The association of pulmonary arterial hypertension and HHT identifies an important disease complication and appears most common among subjects with defects in ALK-1 receptor signalling. Future studies should focus on detailed molecular analysis of the common cellular pathways disrupted by mutations of ALK-1 and BMPR2 that cause inherited pulmonary vascular disease.

Factors determining the post-operative hypo-albuminaemia in cancer patients--stepwise regression analysis.

AIM: To investigate factors influencing post-operative hypo-albuminaemia in cancer patients, with special reference to low dose albumin and/or immunoglobulins administration. PATIENTS AND METHODS: In 270 patients with malignant neoplasms, who underwent extensive chest and/or abdominal surgery, albumin concentrations on the first four postoperative days were examined. One hundred and three high-risk patients received human immunoglobulins intravenously; 44 were given albumin. Univariate and multivariate regression analyses were used to determine the factors influencing albuminaemia on the first four postoperative days. RESULTS: Mean nadir of hypo-albuminaemia occurred on the third postoperative day. In the multivariate analysis, a positive correlation was found between postoperative albuminaemia and pre-operative albuminaemia (b = 0.4919; p = 0.0000) as well as male gender (b = 2.0939; p = 0.0025). A negative correlation was found with the duration of surgery (b = -0.0416; p = 0.0212), pre-operative plasma protein (b = -0.2118, p = 0.0130) and postoperative immunoglobulin administration (b = -1.8858, p = 0.0074). CONCLUSIONS: Postoperative albuminaemia is positively correlated with pre-operative albuminaemia and male gender and negatively correlated with the duration of surgery, pre-operative proteinaemia and postoperative Ig administration.

Enzymatic and histological alterations in the isolated perfused rat pancreas under conditions of oxidative stress.

BACKGROUND: Oxidative stress is a relevant event in the pathogenesis of acute pancreatitis. Investigations in vivo are limited because of the complexity of the organism and the short half-life of free radicals. The isolated perfused rat pancreas could be useful for investigations in the early phase of acute pancreatitis especially under conditions of oxidative stress. METHODS: External perfusions of the pancreatic glands of Wistar rats were carried out using a modified Krebs-Ringer buffer including an additive of the detergents Triton X-100 and a perfusion including hydrogen peroxide (0.0012%) or tert-butylhydroperoxide (0.0042%) or xanthine oxidase (0.1 U/ml). Changes in amylase, lipase, LDH in the portal outflow fluid and for histological alterations were analyzed. RESULTS: Damage to pancreatic parenchyma using Triton X-100 was indicated by increased levels of pancreatic enzymes in the perfusion medium. During perfusion with hydrogen peroxide or tert-butylhydroperoxide we found no changes in pancreatic enzymes in the portal outflow. In contrast, perfusion with xanthine oxidase induced a significant elevation in lipase and amylase in the effusion fluid after 30 min. We found a significant increase in edema in the hydrogen peroxide and in the xanthine oxidase group. Focal necroses of the pancreatic parenchyma were detected in all groups of oxidative stress. CONCLUSIONS: The isolated perfused rat pancreas is a valuable experimental model for investigating the early phase of pathophysiology in acute pancreatitis, for instance, the effect of oxidative stress as an early event in acute pancreatitis. Using hydrogen peroxide tert-butylhydroperoxide or xanthine oxidase, only xanthine oxidase was able to induce a typical elevation in the pancreas enzymes in the effusion fluid.

[The isolated perfused rat pancreas - an experimental model for investigation the early events in the pathogenesis of acute pancreatitis]. / Die isolierte extrakorporale Perfusion des Rattenpankreas - Ein Modell zur Untersuchung der Pathophysiologie der akuten Pankreatitis.

METHODS: External perfusions of the pancreatic glands of Wistar-rats were done, using a modified Krebs-Ringer-Buffer (KRB). We looked for an elevation of amylase, lipase and lactate-dehydrogenase in the effusion fluid (portal outflow fluid). We investigated a normal perfusion (KRB, 60 minutes), a long term perfusion (KRB, 240 minutes) and a perfusion (60 minutes) including an additive of the detergents triton x-100 or the cholecystokinin analogue ceruletid (10(-8) M). RESULTS: An isolated external perfusion of a rat pancreas is possible without inducing any increase of parameters of damage such as amylase, lipase or lactate-dehydrogenase in the outflow medium. The perfusion time should be limited to 80 minutes including a 20 minutes equilibration period. A damage of pancreatic parenchyma is indicated by increased levels of pancreatic enzymes in the perfusion medium. Such damage can be induced by various noxious substances like detergents or cerulein, which has a significance in the pathophysiology of experimental acute pancreatitis. A significant increase (p < 0.01) of lactate-dehydrogenase, lipase and amylase was found 10, 20 and 30 minutes after an application of triton x-100. During a perfusion with the cholecystokinin analogue ceruletid (10(-8) M) we found an increase of lipase (p < 0.05) after 30 minutes and an increase of amylase (p < 0.05) after 50 minutes perfusion. CONCLUSIONS: The isolated perfused rat pancreas is a valuable experimental model to investigate the early phase of pathophysiology in acute pancreatitis.

[Enzymatic and histological alterations in the isolated perfused rat pancreas in the taurocholate and cerulein model of acute pancreatitis]. / Enzymatische und histologische Veränderungen am isoliert perfundierten Pankreas der Ratte im Taurocholat- und Cerulein-Modell der akuten Pankreatitis.

METHODS: The pancreas of 24 male Wistar rats was perfused extracoporally by modified Krebs-Ringer-buffer for 80 minutes (including a 20 minutes equilibration period). To verify any organ damage we measured the activity of pancreatic enzymes like amylase, lipase and lactatdehydrogenase in the portal effluent. Furthermore histological changes were analysed after perfusion. Organ damage was induced by adding cerulein in a physiological dose (10(-10) M, n = 6) and in a supramaximal dose (10(-8) M, n = 6) and by intraductal injection of taurocholate (3.5 %, n = 6). RESULTS: Already 10 minutes after stimulation with cerulein (10(-8) M) and after intraductal injection of taurocholate increased activities (p < 0.01) of amylase and lipase were measured in the portal effluent compared to the group without any treatment. Lactatdehydrogenase levels did not changed. Apart from marked oedema in both groups considerable zones of necrosis could be noticed especially in the taurocholate group. CONCLUSION: Our data suggest that the isolated perfused rat pancreas (IPRP) is a valuable experimental tool to verify pathophysiological changes in the early phase of acute pancreatitis (AP). Various established models of AP such as by cerulein hyperstimulation or intraductal injection of taurocholate, could be applied to the IPRP. We conclude that this method enlarges the spectrum of established experimental models of acute pancreatitis.

Preparation, structure, and reactivity of Ge-containing heterometallic cube derivatives of [M(3)E(4)(H2O)(9)](4+) (M = Mo, W; E = S, Se).

Studies leading to the incorporation of Group 14 germanium into the incomplete cuboidal clusters [M(3)E(4)(H2O)(9)](4+) (M = Mo, W; E = S, Se) have been carried out. From the clusters [Mo(3)E(4)(H2O)(9)](4+), corner-shared double cubes [Mo(6)GeE(8)(H2O)(18)] are obtained with GeO, by heating with Ge powder at 90 degrees C, or by heating with GeO(2) in the presence of H(3)PO(2) as reductant at 90 degrees C, illustrating the dominance of the double cubes. The yellow-green single cube [Mo(3)GeS(4) (H2O)(12)](6+) is only obtained by controlled air oxidation of [Mo(6)GeS(8)(H2O)(18)](8+) over a period of approximately 4 days followed by Dowex purification. In the case of the trinuclear clusters [W(3)E(4)(H2O)(9)](4+), the single cubes [W(3)GeE(4)(H2O)(12)](6+) are dominant and prepared by the reactions with GeO, or GeO(2)/H(3)PO(2). Conversion of [W(3)GeE(4)(H2O)(12)](6+) to the corresponding double cubes is achieved by reductive addition with BH(4)(-) in the presence of a further equivalent of [W(3)E(4)(H2O)(9)](4+). The crystal structures (pts(-) = p-toluene-sulfonate) of [Mo(6)GeS(8)(H2O)(18)](pts)(8).28H2O, (1); [W(6)GeS(8)(H2O)(18)](pts)(8).23H2O, (2); and [Mo(6)GeSe(8)(H2O)(18)](pts)(8).8H2O, (3); have been determined, of which (2) is the first structure of a W(6) double cube. The M-M bond lengths of approximately 2.7 A are consistent with metal-metal bonding, and the M-Ge of approximately 3.5 A corresponds to nonbonding separations. Of the Group 13-15 corner-shared double cubes from [Mo(3)S(4)(H2O)(9)](4+), [Mo(6)GeS(8)(H2O)(18)](8+) is the least reactive with [Co(dipic)(2)](-) as oxidant (0.077 M(-1) s(-1)), and [Mo(6)SnS(8)(H2O)(18)](8+) is next (14.9 M(-1) s(-1)). Both Ge and Sn (Group 14) have an even number of electrons, resulting in greater stability. In contrast, [W(6)GeS(8)(H2O)(18)](8+) is much more reactive (7.3 x 10(3) M(-1) s(-1)), and also reacts more rapidly with O(2).

A capillary electrophoretic system with an improved gas-phase chemiluminescence nitrogen detector.

A new, rugged nebulizer interface was designed to couple a capillary electrophoretic system to a gas-phase nitrogen chemiluminescence detector. By hydraulically decoupling the separation capillary from the nebulizer of the gas-phase nitrogen chemiluminescence detector, the interface-related loss of separation efficiency could be limited to a mere 10%. The ozone reaction chamber of the gas-phase nitrogen chemiluminescence detector has been redesigned leading to a threefold improvement in detection sensitivity over the old design. In combination with a field-amplified sample stacking procedure, the new system was used to detect 0.25 ppm concentrations of UV chromophore-free aminoglycosides.

Distribution of dissolved and labile particulate trace metals in the overlying bottom water in the Vistula River plume (southern Baltic Sea).

Overlying bottom water samples were collected in the Vistula River plume, southern Baltic Sea, (Poland) and analysed for dissolved and labile particulate (1 M HCl extractable) Cu, Pb, Zn, Mn, Fe and Ni, hydrological parameters being measured simultaneously. Particulate organic matter (POM), chlorophyll a and dissolved oxygen are key factors governing the chemical behaviour of the measured metal fractions. For the dissolved Cu, Pb, Zn, Fe and Ni two maxima, in the shallow and in the deeper part of the river plume, were found. In the shallow zone desorption from seaward fluxing metal-rich riverine particles account for markedly increased metal concentrations, as confirmed also by high particulate metal contents. For Pb, atmospheric inputs were also considered to have contributed to the elevated concentrations of dissolved Pb adjacent to the river mouth. In the deep zone desorption from detrital and/or resuspended particles by aerobic decomposition of organic material may be the main mechanism responsible for enrichment of particle-reactive metals (Cu, Pb, Zn) in the overyling bottom waters. The increased concentrations of dissolved Fe may have been due to reductive dissolution of Fe oxyhydroxides within the deep sediments by which dissolved Ni was released to the water. The distribution of Mn was related to dissolved oxygen concentrations, indicating that Mn is released to the water column under oxygen reduced conditions. However, Mn transfer to the dissolved phase from anoxic sediments in deeper part of the Vistula plume was hardly evidenced suggesting that benthic flux of Mn occurs under more severe reductive regime than is consistent with mobilization of Fe. Behaviour of Mn in a shallower part has been presumably affected by release from porewaters and by oxidization into less soluble species resulting in seasonal removal of this metal (e.g. in April) from the dissolved phase. The particulate fractions represented from about 6% (Ni) and 33% (Mn, Zn, Cu) to 80% (Fe) and 89% (Pb) of the total (labile particulate plus dissolved) concentrations. The affinity of the metals for particulate matter decreased in the following order: Pb > Fe > Zn > or = > Cu > Mn > Ni. Significant relationships between particulate Pb-Zn-Cu reflected the affinity of these metals for organic matter, and the significant relationship between Ni-Fe reflected the adsorption of Ni onto Fe-Mn oxyhydroxides. A comparison of metal concentrations with data from other similar areas revealed that the river plume is somewhat contaminated with Cu, Pb and Zn which is in agreement with previous findings on anthropogenic origin of these metals in the Polish zone of southern Baltic Sea.

MC1288, a vitamin D analog, prevents acute graft-versus-host disease in rat bone marrow transplantation.

The major obstacle to successful bone marrow transplantation (BMT) is graft-versus-host disease (GVHD). Vitamin D analogs have shown their efficacy in solid organ transplantation. The purpose of this study was to investigate the suitability of a novel vitamin D analog, MC1288, in the prevention of acute GVHD in a rat BMT model. Allogeneic BMT were performed from Lewis to BN rats (n = 18). The animals were divided into four groups: an untreated control group, MC1288, cyclosporin A (CsA), and MC1288 + CsA-treated groups. Rats were harvested for histology and immunohistochemistry on day 20 after BMT. Histological changes for GVHD in liver, skin, and spleen were scored. Positivity in immunostaining was quantified as the number of positive cells/high power field. Treatment with MC1288 decreased clinical signs of GVHD compared with untreated or CsA-treated rats. Histological manifestations of GVHD, expressed as mean total increment, were significantly lower (1.4 +/- 0.5) in MC1288 than in untreated (5.0 +/- 1.6) or CsA (3.5 +/- 1.0) groups. Combining MC1288 and CsA further improved histology (1.1 +/- 0.6). The expression of CD4, CD8, MHC class II, interleukin-2 receptor, nitric oxide 2, and NKR-P1A (NK cells) positivity was significantly decreased in the liver and skin of BMT rats by MC1288. MC1288 was effective in preventing clinical and histological signs and symptoms of GVHD. This novel vitamin D analog could be used as an immunomodulating agent in BMT.

Induction and expression of cyclin D3 in human pancreatic cancer.

PURPOSE: Cyclins play a key role in the control and regulation of the cell cycle. The role of cyclins in the pathogenesis of pancreatic cancer is largely unknown. METHODS: Using Northern blot analysis, polymerase chain reaction (PCR) and immunohistochemistry, we examined the expression of cyclins D1, D2, and D3 in human pancreatic cancer and studied the induction of these cyclins by growth factors in pancreatic cancer cell lines. RESULTS: We now report that cyclin D1 and D3 mRNAs are expressed in human pancreatic cancer cell lines, and that the expression of cyclin D3 is enhanced in pancreatic cancer cells by amphiregulin, a member of the epidermal growth factor family. Cyclins D1 and D3 are also expressed in normal and malignant pancreatic tissues. However, while the normal pancreas and pancreatic cancers express cyclin D2 as determined by reverse-transcriptase PCR, we could not detect cyclin D2 mRNA by either Northern blot analysis or reverse transcriptase PCR in the two pancreatic cancer cell lines. Immunohistochemical analysis revealed the expression of cyclin D3 in pancreatic cancer cells. CONCLUSIONS: These findings suggest that D-type cyclins are differentially expressed in pancreatic cancer and that the aberrant activation of the EGF receptor in human pancreatic cancer by amphiregulin may lead to the progression of the cell cycle via induction of cyclin D3 expression, thus contributing to the growth advantage of these transformed cells.

Thermodynamic, kinetic and pH studies on the reactions of NCS-, N3-, and CH3CO2- with Fusarium galactose oxidase.

Thermodynamic and kinetic studies on the X- = NCS-, N3-, and CH3CO2- replacement of H2O/OH- at the CuII exogenous site of the tyrosyl-radical-containing enzyme galactose oxidase (GOaseox) from Fusarium (NRR 2903), have been studied by methods involving UV-vis spectrophotometry (25 degrees C), pH range 5.5-8.7, I = 0.100 M (NaCl). In the case of N3- and CH3CO2- previous X-ray structures have confirmed coordination at the exogenous H2O/OH- site. From the effect of pH on the UV-vis spectrum of GOaseox under buffer-free conditions, acid dissociation constants of 5.7 (pK1a; coordinated H2O) and 7.0 (pK2a; H+Tyr-495) have been determined. At pH 7.0 formation constants K(25 degrees C)/M-1 are NCS- (480), N3- (1.98 x 10(4)), and CH3CO2- (104), and from the variations in K with pH the same two pKa values are seen to apply. No pK1a is observed when X- is coordinated. From equilibration stopped-flow studies rate constants at pH 7.0 for the formation reaction kf(25 degrees C)/M-1 s-1 are NCS- (1.13 x 10(4)) and N3- (5.2 x 10(5)). Both K and kf decrease with increasing pH, consistent with the electrostatic effect of replacing H2O by OH-. In the case of the GOaseox Tyr495Phe variant pK1a is again 5.7, but no pK2a is observed, confirming the latter as acid dissociation of protonated Tyr-495. At pH 7.0, K for the reaction of four-coordinate GOaseox Tyr495Phe with NCS- (1.02 x 10(5) M-1) is more favorable than the value for GOaseox. Effects of H+Tyr-495 deprotonation on K are smaller than those for the H2O/OH- change. The pK1a for GOasesemi is very similar (5.6) to that for GOaseox (both at CuII), but pK2a is 8.0. At pH 7.0 values of K for GOasesemi are NCS- (270 M-1), N3- (4.9 x 10(3)), and CH3CO2- (107).