Photobehavior of (alpha-diimine)dimesitylplatinum(II) complexes.

The photobehavior of complexes of the type Pt(diimine)(mes)2 is investigated (where diimine = 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (tmp), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), and 4,7-diphenyl-1,10-phenanthroline (dpp) and mes = the mesityl (2,4,6-trimethylphenyl) anion). For all compounds studied, solution RT emission is observed to be weak and excited-state lifetimes are found to be short (< or = 20 ns) regardless of solvent choice. Evidence is presented for energy-transfer quenching of Pt(dpp)(mes)2 luminescence in toluene by dissolved O2 (primarily producing singlet oxygen) with an observed quenching rate constant of kq > or = 1.3 x 10(9) M-1 s-1. Electron-transfer quenching is also observed in the presence of 3,5-dinitrobenzonitrile, yielding a quenching rate constant of kq > or = 1.6 x 10(9) M-1 s-1. The latter observation suggests that phase Pt(II) systems may have future value as excited-state reductants. All of the complexes display a much more intense and longer-lived luminescence in the solid state at room temperature. Several possible explanations for this dependence on phase are proposed, with the most probable mechanism involving radiationless deactivation in solution via rotation of the o-methyl groups of the mesityl ligands.

Overexpression of cyclooxygenase-2 induces cell cycle arrest. Evidence for a prostaglandin-independent mechanism.

The immediate-early gene cyclooxygenase 2 (Cox-2) is induced in a variety of hyperplastic pathological conditions, including rheumatoid arthritis and colorectal cancer. Although a causal role for Cox-2 has been proposed, mechanisms by which Cox-2 function contributes to the pathogenesis of hyperplastic disease are not well defined. We constructed a green fluorescent protein-tagged Cox-2 (Cox-2-GFP) to examine its effects on a variety of cell types upon overexpression. Subcellular localization and enzymatic and pharmacological properties of Cox-2-GFP polypeptide were indistinguishable from those of the wild-type Cox-2 polypeptide. Overexpression of the Cox-2-GFP or the Cox-2 polypeptide by transient transfection suppressed the population of cells in the S phase of the cell cycle, with a concomitant increase in G(0)/G(1) population. In contrast, transient overexpression of GFP had no effect on cell cycle distribution, whereas endoplasmic reticulum-retained GFP (GFP-KDEL) overexpression was associated with only a minor decrease of cells in S phase. Interestingly, neither NS-398 (a Cox-2-specific inhibitor) nor indomethacin could reverse the effect of Cox-2-GFP overexpression on cell cycle progression. Furthermore, two mutants of Cox-2, S516Q and S516M, which lack the cyclooxygenase activity, exhibited the same effect as Cox-2-GFP. The cell cycle effect of Cox-2-GFP was observed in ECV-304, NIH 3T3, COS-7, bovine microvascular endothelial cells, and human embryonic kidney 293 cells. These findings suggest that Cox-2 inhibits cell cycle progression in a variety of cell types by a novel mechanism that does not require the synthesis of prostaglandins.

Sphingosine-1-phosphate: extracellular mediator or intracellular second messenger?

Sphingosine-1-phosphate (SPP), a polar sphingolipid metabolite, has received much attention recently as an extracellular mediator and an intracellular second messenger. It regulates a wide range of biological responses such as cell growth, death, differentiation, and migration. Recent identification of plasma membrane receptors and the cloning of SPP metabolizing enzymes have increased our understanding of the biology of SPP synthesis and action. However, controversy exists regarding the mode of action of this molecule. EDG-1 and related G-protein-coupled receptors were identified recently as plasma membrane receptors for SPP. In light of this recent discovery, many of the functions of SPP previously thought to be due to intracellular second messenger action should be reevaluated. In addition, signaling properties and functions of the three known receptors for SPP need to be fully delineated. The structures and the evolutionary conservation of SPP metabolizing enzymes from yeast to mammals support the hypothesis that SPP also plays a role as an intracellular second messenger. However, definitive assignment of the intracellular role of SPP awaits purification/molecular cloning of elusive intracellular receptors. Better knowledge of the molecular basis of SPP action is needed to assess the physiological and pathophysiological significance of this bioactive lipid mediator.

Lysophosphatidic acid stimulates the G-protein-coupled receptor EDG-1 as a low affinity agonist.

EDG-1, an inducible G-protein-coupled receptor from vascular endothelial cells, is a high affinity receptor for sphingosine 1-phosphate (SPP) (Lee, M-J., van Brocklyn, J. R., Thangada, S., Liu, C. H., Hand, A. R., Menzeleev, R., Spiegel, S., and Hla, T. (1998) Science 279, 1552-1555). In this study, we show that lysophosphatidic acid (LPA), a platelet-derived bioactive lipid structurally related to SPP, is an agonist for EDG-1. LPA binds to EDG-1 receptor with an apparent Kd of 2.3 microM. In addition, LPA binding to EDG-1 induces receptor phosphorylation, mitogen-activated protein kinase activation, as well as Rho-dependent morphogenesis and P-cadherin expression. These data suggest that LPA is a low-affinity agonist for EDG-1. Activation of the endothelial receptor EDG-1 by platelet-derived lipids LPA and SPP may be important in thrombosis and angiogenesis, conditions in which critical platelet-endothelial interactions occur.

Thermally induced DNA.RNA hybrid to G-quadruplex transitions: possible implications for telomere synthesis by telomerase.

Telomerase is a specialized reverse transcriptase that contains its own RNA template for synthesis of telomeric DNA [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337; Shippen-Lentz, D., & Blackburn, E. H. (1990) Science 247, 546-552]. The activity of this ribonucleoprotein enzyme has been associated with cancer cells [Kim et al. (1994) Science 266, 2011-2015] and is thus a potential target for anticancer chemotherapy. Telomeric DNA.RNA hybrids are important intermediates in telomerase function and form after extension of the growing telomere on the telomerase RNA template. Translocation is a critical step in telomerase function and consists of unwinding of the telomeric DNA.telomerase RNA hybrid followed by repositioning of the 3'-end of the extended telomere. A central question in telomerase function is how translocation of the extended telomere occurs in the absence of ATP or GTP. It has been hypothesized that unwinding of the telomeric hybrid may be facilitated by the formation of stable hairpins or G-quadruplexes by the telomere product (i.e., a hybrid to G-quadruplex transition) and that this may provide at least part of the driving force for translocation [Shippen-Lentz & Blackburn, 1990; Zahler et al. (1991) Nature 350, 718-720]. However, so far there has been no effort aimed at examining the possibility that a hybrid/G-quadruplex equilibrium can occur and to what extent this equilibrium depends on buffer and concentration conditions. Examination of these transitions may provide insight into telomerase function and may also provide clues for the development of anti-telomerase agents. Using a model system consisting of the DNA.RNA hybrid d(GGTTAGGGTTAG).r(cuaacccuaacc), we present evidence that a thermally induced transition of telomeric DNA.RNA hybrid to G-quadruplex can occur under certain conditions. These results provide support for the hypothesis that G-quadruplex formation by the telomere product may in fact regulate telomerase function at the translocation step (Zahler et al., 1991) and suggest an Achilles' heel for indirectly targeting telomerase. Thus, on the basis of the insight gained from the present studies and the results of Zahler et al. (1991), we propose that ligands that selectively bind or cleave G-quadruplex structures may modulate telomerase processivity.

Identification of uterine-related sympathetic neurons in the rat inferior mesenteric ganglion: neurotransmitter content and afferent input.

The rat uterus is innervated by sensory and autonomic nerves. Sensory and sympathetic fibers travel in the hypogastric nerves and are associated with the thoracolumbar spinal cord levels T13-L3. The inferior mesenteric ganglion (IMG) contains the somata of sympathetic postganglionic neurons and some of these may project axons to the uterus. Sensory and parasympathetic fibers travel in the pelvic nerve and are associated with the lumbosacral cord levels L6-S1 and pelvic ganglion (PG). We previously reported data concerning the neurochemical anatomy of the PG with regard to the uterine innervation; the present study was undertaken to characterize the neurochemical anatomy of the IMG with regard to it involvement in uterine innervation. A retrograde axonal tracer was used to verify projections of axons of IMG neurons to the uterus. Immunostaining of cryostat sections of the IMG revealed neurons immunoreactive for neuropeptide Y (NPY) and for tyrosine hydroxylase (TH). Immunostaining for the synaptic terminal protein synapsin I (SYN) revealed numerous fine terminals immediately surrounding the principal neurons and in the interneuronal spaces. Varicosities immunoreactive for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), substance P (SP) and galanin (GAL) appear to be associated with principal neurons. Additional varicosities stained for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and nitric oxide synthase (NOS), thus indicating sites of neuronal nitric oxide synthesis. This study revealed that the IMG contains uterine-related neurons and that some of the retrogradely labeled uterine-related neurons contain NPY, TH or both NPY/TH. In addition, uterine-related neurons received abundant afferent inputs indicated by SYN-immunoreactive (-ir) terminals and some of these varicosities labeled for GAL, CGRP, VIP, ENK, or NADPH-d/NOS.

Quantitation of ultraviolet radiation-induced cyclobutyl pyrimidine dimers in DNA by video and photographic densitometry.

We have compared video and photographic methods for calculating the number of ultraviolet radiation (uv)-induced pyrimidine dimers in DNA from the bacteriophage T7 exposed to uv (0 to 800 J/m2) from an FS40 sunlamp. DNA was incubated with a pyrimidine dimer-specific Micrococcus luteus uv endonuclease, subjected to alkaline agarose gel electrophoresis, neutralized, and stained with ethidium bromide, and the DNA fluorescence was recorded either with a video camera or on photographic film. The slopes of the dose-response curves for the number of uv-endonuclease-sensitive sites per 10(3) bases (pyrimidine dimers) was 1.2 (+/- 0.1) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the video analysis and 1.3 (+/- 0.04) X 10(-4) uv-endonuclease-sensitive sites per J/m2 for the photographic analysis. Results for pyrimidine dimer determination by either method were statistically comparable.

Electrophoretic separation of nucleic acids: evaluation by video and photographic densitometry.

The separation of DNA by gel electrophoresis provides a rapid method for determining size distributions of DNA in solution. Densitometric scanning of photographs of gels has been the standard method of analysis of agarose gels. However, analysis of photographs is complicated by the non-linear response of photographic film. Charged-coupled device video cameras have become popular for quantitative densitometry and we have used a charge-coupled device camera to image agarose gels to quantitate DNA damage. We compare video and photographic densitometry for quantitation of ultraviolet radiation (UV)-induced DNA damage and find that the two methods give equivalent results.

Evaluation of densitometry data using interactive computer graphics: application to DNA agarose gels.

A method of analyzing DNA agarose gels using interactive computer graphics is described. After electrophoresis in an alkaline agarose gel, DNA is neutralized, stained with ethidium bromide and excited with ultraviolet radiation. The resulting fluorescent distribution on the gel is photographed, and the negative scanned by a digitizing densitometer. The data is subsequently analyzed using a computer program developed to facilitate manipulation and selection of data from the densitometer trace. The method has been applied to determine pyrimidine dimer yields in DNA from human lymphocytes exposed to UV radiation. The technique significantly reduces the time required to analyze such data, while also providing greater accuracy. The method could be easily adapted to assist in similar analyses of other macromolecules such as RNA or proteins.

Is maternal alpha-fetoprotein screening still of value in a low-risk area for neural tube defects?

Estimation of maternal serum alpha-fetoprotein (AFP) was used as a screening method for the detection of neural tube defects (NTDs) in 6344 women over three years. Of 88 (1.4 per cent) who had one or more serum AFP levels equal to, or greater than, 2.5 multiples of the median (MoM) for the relevant gestational age, 43 (0.68 per cent) underwent amniocentesis. There were eight NTDs. Four of these were screened by serum AFP, and all cases of spina bifida had serum AFP levels greater than 3.0 MoM, including one small open defect which was not seen on ultrasound. The other four cases of NTD, which were not screened, were identified by ultrasound. Of 64 singleton pregnancies 32 (50 per cent) had serum AFP levels between 2.5 and 3.0 MoM, and low birthweight (less than or equal to 2500 g) occurred in 29 per cent. Because of improvements in ultrasound techniques and the apparent falling incidence of NTD, the role of serum AFP as the primary screening procedure should be regularly reviewed. Effective screening is dependent on mothers booking early.

A simple and inexpensive method for assessing in vitro candidacidal activity of leukocytes.

Candidacidal activity of mouse neutrophils and macrophages was determined directly in microtiter plates. After a suitable period of interaction between phagocytic cells and C. albicans in the wells, the mouse cells were lysed with distilled water and corn meal agar was added to each well. Following incubation at 37 degrees C, viability was assessed using an inverted microscope and counting the number of germ tubes or microcolonies which developed. This method does not use radioisotopes or vital stains and should be applicable to other genera of yeasts.

Comparison of binding of bovine and human thyroid-stimulating hormone to receptor sites on human thyroid membranes.

The binding of 125I-labelled bovine TSH (bTSH) to a wide range of human thyroid membrane preparations was compared with that of 125I-labelled human TSH (hTSH). Much higher binding percentages were obtained with the 125I-labelled bTSH. This was because the receptors had a higher binding affinity for bTSH than for hTSH. No differences in tracer purity, nor differences in optimal conditions for the binding of bTSH or hTSH, nor tracer degradation contributed significantly to the better binding of 125I-labelled bTSH. Good correlation was found between binding percentages for 125I-labelled bTSH and 125I-labelled hTSH over the range of thyroid specimens. Useful information on human TSH receptors is, therefore, obtainable from binding studies with 125I-labelled bTSH. The TSH displacement curves yielded linear Scatchard plots whenever the tracer and displacing hormones were of the same species. The data were, therefore, consistent with a simple binding reaction between TSH and a single set of independent receptor sites.

Absorption by the colon during prolonged infusions in conscious, unrestrained rats.

1. Colonic absorption was studied in conscious, unrestrained rats during prolonged infusions through implanted cannulae. During infusion of predominantly NaCl-containing solution at rates up to 0.7 ml h-1 per 100 g body wt., absorption of fluid and Na increased considerably without significant change of transmucosal p.d.; diarrhoea did not occur. 2. Exclusion of the distal colon by colostomy showed that the proximal part of the colon was chiefly responsible for the increased absorption and inspection during the infusion showed it to be much dilated. Removal of the caecum showed that it contributed considerably to absorption by the proximal part. The distal colon influenced Na concentration in the faeces but absorbed little volume while direct infusion into this region rapidly produced diarrhoea. 3. The addition of Na deoxycholate (5 mmol/l) to the infusion solution impaired absorption provoking diarrhoea with mucus loss; there was no evidence that fluid secretion was stimulated. Substitution of SO4 for Cl in the infused solution produced only small changes of transmucosal p.d. but considerably impaired absorption and diarrhoea resulted. 4. The findings indicate that the proximal colon and caecum possess a considerable potential for increasing fluid and Na absorption and suggest the possibility that a neutral Na-Cl coupled absorptive mechanism is stimulated by loading the colon with fluid and NaCl.

Iodination of thyroid-stimulating hormone for receptor-binding studies with human thyroid membranes: effects of specific activity and method of iodination.

Three different methods were compared for 125I-labelling of thyroid-stimulating hormone (TSH) for use in receptor-binding studies with human thyroid membranes: these were the chloramine-T, Bolton-Hunter and lactoperoxidase methods. Chloramine-T proved to be an inferior method to the other two. Iodinations to different specific activity (0.7--9.4 Bq/pg) were also compared: too high a specific activity led to reduced binding and a dramatic shift in the pH optimum for the TSH-receptor interaction. A specific acitivity of 2.5 Bq/pg should not be exceeded if binding of 125I-labelled TSH is to be representative of the binding of the natural hormone. Under these conditions, pH 7.5 was optimal for binding of TSH to its receptor. Repurification of the labelled TSH by receptor adsorption also proved to be essential.

Prolonged intravenous infusion of labelled iodocompounds in the rat: [125I]iodide metabolism and effects of moderate dietary iodine deficiency.

Radio-iodide was administered by prolonged continuous intravenous infusion to rats maintained under iodine-replete conditions and in moderate iodine deficiency. A close approximation to equilibrium labelling was thereby achieved. Labelled iodocompounds extracted from various tissues were analysed by thin-layer chromatography. Moderate iodine deficiency resulted in a slight increase in the ratio of mono-iodotyrosine to di-iodotyrosine in the thyroid. No change in the ratio of tri-iodothyronine (T3) to thyroxine (T4) was found in thyroid, plasma or skeletal muscle. Faecal excretion of T3 declined appreciably relative to that of T4. Under iodine-replete conditions the ratio of thyroidal secretion rates of T3 and T4 was estimated to be more than three times higher than the ratio of these iodocompounds within the thyroid. Heterogeneity of thyroglobulin structure and function may explain these observations.

Prolonged intravenous infusion of labelled iodocompounds in the rat: [125I]thyroxine and [125I]tri-iodothyronine metabolism and extrathyroidal conversion of thyroxine to tri-iodothyronine.

Extrathyroidal tissues of the rat were labelled to steady state by prolonged continuous intravenous infusion of 125I-labelled thyroxine (T4) or tri-iodothyronine (T3). Labelled iodocompounds extracted from various tissues were analysed by thin-layer chromatography. Signifficant amounts of labelled T3 were found in all tissues examined after infusion of [125I]T4, confirming that conversion of T4 to T3 occurs in extrathyroidal tissues of the rat. Faecal excretion of labelled T3 after [125I]T4 infusion provided an assessment of the extent of extrathyroidal conversion: about a third of the T4 was metabolized by this pathway. Extrathyroidal conversion was independently estimated to account for about a third of the total production of T3. The site of extrathyroidal conversion was established by comparing the distribution of labelled T3 after the two types of infusion: kidney and liver were both prominent sites of conversion of T4 to T3.