Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

Docetaxel, cisplatin, and fluorouracil; docetaxel and cisplatin; and epirubicin, cisplatin, and fluorouracil as systemic treatment for advanced gastric carcinoma: a randomized phase II trial of the Swiss Group for Clinical Cancer Research

This randomized phase II trial evaluated two docetaxel-based regimens to see which would be most promising according to overall response rate (ORR) for comparison in a phase III trial with epirubicin-cisplatin-fluorouracil (ECF) as first-line advanced gastric cancer therapy.

Synthesis of the bicyclo-[4.3.0]thymidyl nucleoside via Pd(II)-mediated ring expansion chemistry

A variety of modified nucleosides to improve antisense oligodeoxynucleotide properties such as target affinity, nuclease resistance, and pharmacokinetics were developed in the last two decades. In the context of conformational restriction we present here the synthesis of the [4.3.0]-bicyclo-DNA thymine monomer via Pd(II)-mediated ring expansion of an intermediate of the tricyclo-DNA synthesis.

Adding cetuximab to capecitabine plus oxaliplatin (XELOX) in first-line treatment of metastatic colorectal cancer: a randomized phase II trial of the Swiss Group for Clinical Cancer Research SAKK

BACKGROUND: To determine the activity and tolerability of adding cetuximab to the oxaliplatin and capecitabine (XELOX) combination in first-line treatment of metastatic colorectal cancer (MCC). PATIENTS AND METHODS: In a multicenter two-arm phase II trial, patients were randomized to receive oxaliplatin 130 mg/m(2) on day 1 and capecitabine 1000 mg/m(2) twice daily on days 1-14 every 3 weeks alone or in combination with standard dose cetuximab. Treatment was limited to a maximum of six cycles. RESULTS: Seventy-four patients with good performance status entered the trial. Objective partial response rates after external review and radiological confirmation were 14% and 41% in the XELOX and in the XELOX + Cetuximab arm, respectively. Stable disease has been observed in 62% and 35% of the patients, with 76% disease control in both arms. Cetuximab led to skin rash in 65% of the patients. The median overall survival was 16.5 months for arm A and 20.5 months for arm B. The median time to progression was 5.8 months for arm A and 7.2 months for arm B. CONCLUSION: Differences in response rates between the treatment arms indicate that cetuximab may improve outcome with XELOX. The correct place of the cetuximab, oxaliplatin and fluoropyrimidine combinations in first-line treatment of MCC has to be assessed in phase III trials.

Reproducibility and accuracy of the ICDAS-II for occlusal caries detection

OBJECTIVES: The aim of this in vitro study was to assess the inter- and intra-examiner reproducibility and the accuracy of the International Caries Detection and Assessment System-II (ICDAS-II) in detecting occlusal caries. METHODS: One hundred and sixty-three molars were independently assessed twice by two experienced dentists using the 0- to 6-graded ICDAS-II. The teeth were histologically prepared and classified using two different histological systems [Ekstrand et al. (1997) Caries Research vol. 31, pp. 224-231; Lussi et al. (1999) Caries Research vol. 33, pp. 261-266] and assessed for caries extension. Sensitivity, specificity, accuracy and area under the ROC curve (A(z)) were obtained at D(2) and D(3) thresholds. Unweighted kappa coefficient was used to assess inter- and intra-examiner reproducibility. RESULTS: For the Ekstrand et al. histological classification the sensitivity was 0.99 and 1.00, specificity 1.00 and 0.69 and accuracy 0.99 and 0.76 at D(2) and D(3), respectively. For the Lussi et al. histological classification the sensitivity was 0.91 and 0.75, specificity 0.47 and 0.62 and accuracy 0.86 and 0.68 at D(2) and D(3), respectively. The A(z) varied from 0.54 to 0.73. The inter- and intra-examiner kappa values were 0.51 and 0.58, respectively. CONCLUSIONS: ICDAS-II presented good reproducibility and accuracy in detecting occlusal caries, especially caries lesions in the outer half of the enamel.

Effects of two traits of the ecological state equation on our understanding of species coexistence and ecosystem services

Species coexistence has been a fundamental issue to understand ecosystem functioning since the beginnings of ecology as a science. The search of a reliable and all-encompassing explanation for this issue has become a complex goal with several apparently opposing trends. On the other side, seemingly unconnected with species coexistence, an ecological state equation based on the inverse correlation between an indicator of dispersal that fits gamma distribution and species diversity has been recently developed. This article explores two factors, whose effects are inconspicuous in such an equation at the first sight, that are used to develop an alternative general theoretical background in order to provide a better understanding of species coexistence. Our main outcomes are: (i) the fit of dispersal and diversity values to gamma distribution is an important factor that promotes species coexistence mainly due to the right-skewed character of gamma distribution; (ii) the opposite correlation between species diversity and dispersal implies that any increase of diversity is equivalent to a route of “ecological cooling” whose maximum limit should be constrained by the influence of the third law of thermodynamics; this is in agreement with the well-known asymptotic trend of diversity values in space and time; (iii) there are plausible empirical and theoretical ways to apply physical principles to explain important ecological processes; (iv) the gap between theoretical and empirical ecology in those cases where species diversity is paradoxically high could be narrowed by a wave model of species coexistence based on the concurrency of local equilibrium states. In such a model, competitive exclusion has a limited but indispensable role in harmonious coexistence with functional redundancy. We analyze several literature references as well as ecological and evolutionary examples that support our approach, reinforcing the meaning equivalence between important physical and ecological principles.

S9511: a Southwest Oncology Group phase II study of trimetrexate, 5-fluorouracil, and leucovorin in unresectable or metastatic adenocarcinoma of the stomach.

OBJECTIVE: The primary objective of this trial was to evaluate the response rate for trimetrexate in conjunction with 5-FU and leucovorin (LV) (= TFL) in the treatment of advanced gastric cancer in a phase II, cooperative group setting. METHODS: Patients with locally advanced, unresectable, or metastatic adenocarcinoma of the stomach received trimetrexate 110 mg/m IV over 60 minutes day 1, followed by 5-FU 500 mg/m IV bolus and LV 200 mg/m IV over 60 minutes day 2, followed by oral LV 15 mg every 6 hours x 7 doses, all weekly for 6 weeks followed by 2 weeks of rest, continued until progression. RESULTS: Characteristics for 37 eligible patients: median age 63 (range: 23-83); male/female: 69% of 31%; performance status 0/1/2 15/20/1. The confirmed response rate was 19%, and median overall survival was 6 months. Two patients died as a result of therapy, 1 because of infection without significant neutropenia, and 1 due to perforation of a responding gastric lesion. Seventy-two percent experienced grades 3 and 4 toxicity, most commonly diarrhea, fatigue, and lymphopenia. CONCLUSIONS: This regimen achieves response rates comparable to other 5-FU-based regimens, when used in treatment of incurable gastric cancer. Toxicity appears manageable.

Interaction of the Trinuclear Triangular Secondary Building Unit [Cu 3 (μ 3 -OH)(μ-pz) 3 ] 2+ with 4,4′-Bipyridine. Structural Characterizations of New Coordination Polymers and Hexanuclear Cu II Clusters. 2°

By reacting 4,4′-bipyridine (bpy) with selected trinuclear triangular CuII complexes, [Cu3(μ3-OH)(μ-pz)3(RCOO)2(LL′)] [pz = pyrazolate anion; R = CH3, CH3CH2, CH2═CH, CH2═C(CH3); L, L′ = Hpz, H2O, MeOH] in MeOH, the substitution of monotopic ligands by ditopic bpy was observed. Depending on the stoichiometric reaction ratios, different compounds were isolated and structurally characterized. One- and two-dimensional coordination polymers (CPs), as well as two hexanuclear CuII clusters were identified. One of the hexanuclear clusters self-assembles into a supramolecular three-dimensional structure, and its crystal packing shows the presence of two intersecting channels, one of which is almost completely occupied by guest bpy, while in the second one guest water molecules are present. This compound also shows a reversible, thermally induced, single-crystal-to-single-crystal transition.

Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis

The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co-ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene.

DESyne novolimus-eluting coronary stent is superior to Endeavor zotarolimus-eluting coronary stent at five-year follow-up: final results of the multicentre EXCELLA II randomised controlled trial

AIMS Newer-generation drug-eluting stents (DES) have been shown to be superior to first-generation DES. Current-generation DES have zotarolimus, everolimus or biolimus as antiproliferative drugs. Novolimus, a metabolite of sirolimus, has been specifically developed to provide efficacy similar to currently available agents at a lower dose and thus requires a lower polymer load. We report the final five-year outcomes of the EXCELLA II trial comparing a zotarolimus-eluting stent (ZES) with a novolimus-eluting stent (NES). METHODS AND RESULTS EXCELLA II is a prospective, multicentre, single-blind, non-inferiority clinical trial. Patients (n=210) with a maximum of two de novo lesions in two different epicardial vessels were randomised (2:1) to treatment with either NES (n=139) or ZES (n=71). At five-year follow-up, patients in the NES group had a significantly lower incidence of the patient-oriented (HR 0.53, 95% CI: 0.32-0.87, p=0.013) and device-oriented (HR 0.38, 95% CI: 0.17-0.83, p=0.011) composite endpoints. There was no difference in cardiac death and definite/probable stent thrombosis between the two groups; however, there was a trend towards reduction in myocardial infarction and repeat revascularisation in the NES group at five-year follow-up. CONCLUSIONS At five-year follow-up, the incidence of device- and patient-oriented events was significantly lower in the NES group. Further studies, adequately powered for clinical outcomes, are warranted. TRIAL REGISTRATION ClinicalTrials.gov number NCT00792753.

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Two independent and primitive envelopes of the bilobate nucleus of comet 67P

The factors shaping cometary nuclei are still largely unknown, but could be the result of concurrent effects of evolutionary(1,2) and primordial processes(3,4). The peculiar bilobed shape of comet 67P/Churyumov-Gerasimenko may be the result of the fusion of two objects that were once separate or the result of a localized excavation by outgassing at the interface between the two lobes(5). Here we report that the comet's major lobe is enveloped by a nearly continuous set of strata, up to 650 metres thick, which are independent of an analogous stratified envelope on the minor lobe. Gravity vectors computed for the two lobes separately are closer to perpendicular to the strata than those calculated for the entire nucleus and adjacent to the neck separating the two lobes. Therefore comet 67P/Churyumov-Gerasimenko is an accreted body of two distinct objects with 'onion-like' stratification, which formed before they merged. We conclude that gentle, low-velocity collisions occurred between two fully formed kilometre-sized cometesimals in the early stages of the Solar System. The notable structural similarities between the two lobes of comet 67P/Churyumov-Gerasimenko indicate that the early-forming cometesimals experienced similar primordial stratified accretion, even though they formed independently.

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight {HSPs} families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the {HSP60} family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.

Structural determinants of subunit -subunit interactions and subcellular localization of the calcium /calmodulin -dependent protein kinase II

The multifunctional Ca$\sp{2+}$/calmodulin-dependent protein kinase II (CaM kinase) is a Ser/Thr directed protein kinase that participates in diverse Ca$\sp{2+}$ signaling pathways in neurons. The function of CaM kinase depends upon the ability of subunits to form oligomers and to interact with other proteins. Oligomerization is required for autophosphorylation which produces significant functional changes that include Ca$\sp{2+}$/calmodulin-independent activity and calmodulin trapping. Associations with other proteins localize CaM kinase to specific substrates and effectors which serves to optimize the efficiency and speed of signal transduction. In this thesis, we investigate the interactions that underlie the appropriate positioning of CaM kinase activity in cells. We demonstrate that the subcellular distribution of CaM kinase is dynamic in hippocampal slices exposed to anoxic/aglycemic insults and to high K$\sp{+}$-induced depolarization. We determine the localization of CaM kinase domains expressed in neurons and PC-12 cells and find that the C-terminal domain of the $\alpha$ subunit is necessary for localization to dendrites. Moreover, monomeric forms of the enzyme gain access to the nucleus. Attempts made to identify novel CaM kinase binding proteins using the yeast two-hybrid system resulted in the isolation of hundreds of positive clones. Those that have been sequenced are identical to CaM kinase isoforms. Finally, we report the discovery of specific regions within the C-terminal domain that are necessary and sufficient for subunit-subunit interactions. Differences between the $\alpha$ and $\beta$ isoforms were discovered that indicate unique structural requirements for oligomerization. A model for how CaM kinase subunits interact to form holoenzymes and how structural heterogeneity might influence CaM kinase function is presented. ^