Novel high-performance liquid chromatographic assay using fluorescence detection for the quantitation of plasma gamma-methylene-10-deazaaminopterin and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin, in patients with solid cancers in a phase I trial

Gamma-methylene-10-deazaaminopterin (MDAM), a unique dihydrofolate reductase inhibitor, has demonstrated antitumor activity against a broad spectrum of human solid tumors in preclinical studies. A novel reversed-phase, ion-pair high-performance liquid chromatography (HPLC) assay that uses fluorescence detection has been developed to quantitate levels of MDAM and its major metabolite, 7-hydroxy-gamma-methylene-10-deazaaminopterin (7-OH-MDAM), in human plasma. The recovery of MDAM and 7-OH-MDAM from plasma was >97% by a simple one-step deproteinization process using tetrabutylammonium bromide (TBABr) and methanol. MDAM and 7-OH-MDAM remained stable in plasma over a 28-day test period at ambient temperatures, and neither compound was light-sensitive. The limit of quantitation was 0.005 microM for both MDAM and 7-OH-MDAM. This assay has been found to be simple, sensitive and reproducible in determining plasma concentrations of MDAM and 7-OH-MDAM in patients with solid cancers in a phase I trial.     

Narratives of Spatial Division： The Role of Social Memory in Shaping Urban Space in Belfast

The paper examines the role of shared spaces in divided cities in promoting future sustainable communities and spaces described as inclusive to all. It addresses the current challenges that prevent such inclusiveness and suggests future trends of its development to be of benefit to the wider city community. It explains how spaces in divided cities are carved up into perceived ownerships and territorialized areas, which increases tension on the shared space between territories; the control of which can often lead to inter-community disputes. The paper reports that common shared space in-between conflicting communities takes on increased importance since the nature of the conflict places emphasis on communities＇ confidence, politically and socially, while also highlighting the necessity for confidence in inclusion and feeling secure in the public domain. In order to achieve sustainable environments, strategies to promote shared spaces require further focus on the significance of everyday dynamics as essential aspects for future integration and conflict resolution.     

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an approximately 50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCepsilon expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCepsilon expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.     

Regulation of cardiac myocyte contractile function by inducible nitric oxide synthase (iNOS): mechanisms of contractile depression by nitric oxide

Inflammatory cytokines have been implicated in the reversible depression of cardiac contractile function accompanying local or systemic immune stimulation. Incubation of cardiac myocytes with soluble components in the supernatant from cultured rat lung macrophages activated with endotoxin decreases their contractile response to beta-adrenergic stimulation through the induction of iNOS and the subsequent production of nitric oxide by these cells. In the present study, we characterize the mechanisms underlying NO's attenuation of adrenergic responsiveness in cardiac myocytes. iNOS was induced in cultured ventricular myocytes from adult rats by incubation for 20 h with conditioned medium from lipopolysaccharide (LPS)-activated macrophages. iNOS induction did not induce any alteration in beta-adrenergic receptor density or affinity, Galphai protein abundance, or adenylyl cyclase activity in cultured myocytes. Myocyte exposure to activated macrophage-conditioned medium markedly attenuated the elevation of cAMP in response to isoproterenol (Iso, 2 nM). Induction of iNOS with the macrophage-conditioned medium also potentiated the Iso-induced increase in myocyte cGMP. This cGMP increase was totally abolished by NOS inhibitors. NOS inhibition also returned the attenuated cAMP response to 2 nM Iso to levels observed in control cells. Pre-incubation of the cells in isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, also partly reversed the attenuation of cAMP increase with 2 nM Iso in cells expressing iNOS. Brief (15 min) exposure of myocytes to the NO donor, S-nitrosoacetylcysteine (SNAC, 100 micro M) which produced a three-fold increase in intracellular cGMP, also decreased by half the contractile response of cardiac myocytes to Iso (2 nM). We conclude that NO endogenously produced by iNOS decreases the intracellular levels of cAMP in response to beta-adrenergic stimulation in isolated cardiac myocytes, in part through a cGMP-mediated mechanism. This effect may participate in the NO-dependent depression of cardiac function following cytokine exposure.     

Cyclooxygenase inhibition reveals synergistic action of vasoconstrictors on mesangial cell growth

Since endogenous vasoconstrictors promote mesangial cell growth and increase the biosynthesis of antiproliferative prostaglandins, the effects of cyclooxygenase inhibition on mesangial cell proliferation should be strongly dependent on the prevailing levels of neuroendocrine vasoconstrictors. We compared the effects of indomethacin (10(-6) M), a cyclooxygenase inhibitor, on [3H]thymidine incorporation by cultured rat mesangial cells in the presence of various combinations of angiotensin II (10(-10) M), [Arg8]vasopressin (10(-11) M), (-)-norepinephrine (10(-8) M) and endothelin-1 (10(-11) M). Indomethacin did not enhance [3H]thymidine incorporation in cells treated with each individual vasoconstrictor, or in cells treated with two-way combinations with the exception of modestly increased [3H]thymidine incorporation in cells treated with angiotensin II + (-)-norepinephrine or [Arg8]vasopressin + (-)-norepinephrine. In contrast, in cells treated with any three-way or the four-way combination, indomethacin markedly increased [3H]thymidine incorporation. Importantly, a highly significant interaction (P<0.0001) was observed for thymidine incorporation between the number of vasoconstrictors present and indomethacin treatment, thus demonstrating that cyclooxygenase inhibition reveals a synergistic action of vasoconstrictors on the DNA synthesis in mesangial cells.     

The etiology of pneumonia in malnourished and well-nourished Gambian children

During a 2-year period 159 malnourished children ages 3 months to 5 years with radiologic evidence of pneumonia were investigated to determine the cause of their pneumonia. In addition 119 malnourished children without pneumonia, 119 well-nourished children with pneumonia and 52 well-nourished children without pneumonia were studied as controls. Percutaneous lung aspiration was performed on 35 malnourished and 59 well-nourished children with pneumonia. Bacteria were isolated from the blood, lung or pleural fluid of 28 (18%) malnourished children with pneumonia, 42 (35%) well-nourished children with pneumonia and from the blood of 5 (4%) malnourished children without pneumonia. Streptococcus pneumoniae and Haemophilus influenzae, which were the two organisms isolated most frequently in both groups of children with pneumonia, were found in 17 (11%) malnourished and 39 (33%) well-nourished children with pneumonia. Mycobacterium tuberculosis was detected in 5 malnourished children with pneumonia. A potentially pathogenic virus was identified in 35% of malnourished children with pneumonia and 40% of well-nourished children with pneumonia, and from 25% of children without pneumonia. The viruses identified most frequently were adenovirus and respiratory syncytial virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo osteogenesis assay: a rapid method for quantitative analysis

A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale scoring method gave an r2=0.767 while the Percentage Bone method gave a value of 0.902. These results indicate that scoring ceramic cubes by the percentage of pores containing bone gives a result that corresponds to image analysis measurements at nearly a 90% confidence level. Thus, the Percentage Bone method of scoring is an accurate and relatively quick scoring method for in vivo bone formation.     

Organism-dependent fungicidal activities of azoles

We investigated the antifungal activities of itraconazole and voriconazole on Aspergillus species by time kill studies, and the results were compared with those obtained for Candida species. Exposure of Aspergillus fumigatus conidia to varying concentrations (1.25 to 10 microg/ml) of itraconazole and voriconazole resulted in cellular death; the cytocidal effect was time and concentration dependent. In contrast, no killing of Candida albicans occurred in the presence of itraconazole and voriconazole at concentrations as high as 10 microg/ml, although candidal growth was inhibited compared to the drug-free control. Amphotericin B (1.25 to 10 microg/ml), on the other hand, killed both A. fumigatus and C. albicans. Similar results were obtained for non-A. fumigatus aspergilli and non-C. albicans Candida species. These observations indicate that both itraconazole and voriconazole are cytocidal agents for Aspergillus species but not for Candida species, suggesting that azoles possess organism-dependent fungicidal activities.