FTIR characterization of heterocycles lumazine and violapterin in solution: effects of solvent on anionic forms

Fourier transform infrared (FTIR) spectra have been obtained from solution samples of the heterocycles uracil, lumazine, and violapterin and reveal interpretable carbonyl stretching frequencies. Spectra of conjugate bases of lumazine and violapterin demonstrate decreases in these carbonyl stretching frequencies upon ionization. Based on isotopic shifts from amide deuterated analogs, semiempirical QCFF/PI calculations were used to assign the vibrational frequencies in the region 1100-1800 cm-1 observed from samples in dimethylsulfoxide (DMSO) and aqueous solutions to specific normal modes. The observed deuterium shifts and the calculations suggest that, in some cases, N-H bending motions are coupled to the C=O stretching motions of the pyrimidine ring. These data suggest that for lumazine anions a change in solvent can significantly change the mixing of the N-H bending and C=O stretching vibrational motions. This implies that vibrational analysis for lumazine species in relatively noninteracting media like nonpolar solvents, mulls or pellets cannot necessarily be transferred to the system when it is dissolved in a polar, hydrogen-bonding solvent such as water. Although other explanations can be offered, our vibrational analysis suggests that the changes in normal mode composition of the predominantly C=O stretching vibrations of lumazine anion on going from dimethylsulfoxide to water solution are consistent with a change in the predominant tautomer of the heterocycle. This change appears to correspond to a shifting of the location of the remaining acidic proton to a different ring nitrogen atom. This interpretation is of interest in view of recent ab initio calculations which suggest that proton shifts may occur during the hydroxylation of lumazine as mediated by the enzyme xanthine oxidase.     

The use of toxicologic data in mechanistic risk assessment: 1,3-butadiene as a case study

The National Research Council (NRC) recently published a report. Science and Judgment in Risk Assessment, that critiqued the current approaches to characterizing human cancer risks from exposure to chemicals. One issue raised in the report relates to the use of default options for quantitation of cancer risks. Default options are general guidelines that can be used for risk assessment when specific information about a chemical is absent. Research on 1,3-butadiene represents an interesting case study in which existing knowledge on this chemical indicates that two default options may no longer be tenable: (1) humans are as sensitive as the most sensitive animal species, and (2) the rate of metabolism is a function of body surface area rather than inherent species differences in metabolic capacity. Butadiene, a major commodity chemical used in the production of synthetic rubber, is listed as one of 189 hazardous air pollutants under the 1990 Clean Air Act Amendments. Butadiene is a carcinogen in rats and mice, with mice being substantially more sensitive than rats. The extent to which butadiene poses a cancer risk to humans exposed to this chemical is uncertain. Butadiene requires metabolic activation to DNA-reactive epoxides to exert its mutagenic and carcinogenic effects. Research is directed toward obtaining a better understanding of the cancer risks of butadiene in humans by evaluating species-dependent differences in the formation of the toxic butadiene epoxide metabolites, epoxybutene and diepoxybutane. The data include in-vitro studies on butadiene metabolism using tissues from humans, rats, and mice as well as experimental data and physiological model predictions for butadiene in blood and butadiene epoxides in blood, lung, and liver after exposure of rats and mice to inhaled butadiene. The findings suggest that humans are more like rats and less like mice regarding the formation of butadiene epoxides. The research approach employed can be a useful strategy for developing mechanistic and toxicokinetic data to supplant default options used in carcinogen risk assessments for butadiene.     

Microsatellite instability in follicle centre cell lymphoma

Fluorescent polymerase chain reaction (PCR) was used to assay 12 microsatellite markers (APC x 2, DCC, P53 x 2, RB1, NM23, WT1, D6S260, D6S262, D6S281 and TNFa) to look for evidence of microsatellite instability in 40 cases of follicle centre cell lymphoma (FCC). Evidence of novel alleles seen in the tumour tissue but not the normal uninvolved tissue was seen in seven cases (17%). In only two of these cases (5%) was more than one locus involved but in these cases multiple affected loci were seen (4/12 and 7/12 respectively). The detection of microsatellite instability indicates a DNA repair defect such as that which would be predicted to occur in cells with mutated mismatch repair genes, a novel finding in FCC lymphoma.     

A cholera toxin-sensitive guanyl nucleotide binding protein mediates the movement of pituitary luteinizing hormone into a releasable pool: loss of this event is associated with the onset of homologous desensitization to gonadotropin-releasing hormone

Cholera toxin (CTX; 5 micrograms/ml), but not pertussis toxin (100 ng/ml), when preincubated with pituitary cells for 18 h, enhances the percentage of cellular LH released in response to continuous or pulsatile administration of 5 x 10(-9) M GnRH. This effect occurs without increasing total (intracellular plus extracellular) LH, indicating that it is best explained by redistribution of LH from a nonreleasable to a releasable pool. This site of action is consistent with the observation that CTX-pretreated cells are also sensitized to stimulation of LH release by the Ca2+ ionophore A23187. The observations that CTX stimulates the production of cAMP in these cells and that the sensitizing action of CTX is mimicked by (Bu)2cAMP (1 mM) are consistent with the view that a CTX-stimulated guanyl nucleotide binding protein, capable of activating adenylyl cyclase, is mediating this sensitization. We used a perifused cell system to show that the movement of LH into a releasable pool is lost with the onset of homologous desensitization due to high pulse frequency or constant administration of GnRH (5 x 10(-9) M, continuous or a pulse each 15 min). Sensitization to CTX is restored by stimulation with a high concentration of GnRH (10(-6) M) or by resetting the pulse frequency to the rate measured in vivo (a pulse each 90 min). Both of these treatments also circumvent the desensitized state, restoring LH release. These results identify a novel lesion associated with the development of desensitization in the gonadotrope and support the role of a CTX-sensitive guanyl nucleotide binding protein in regulation of pituitary responsiveness to GnRH.     

Identification of nonproliferating but viable hypoxic tumor cells in vivo

We have used the combination of pimonidazole labeling of hypoxic cells, bromodeoxyuridine labeling of proliferating cells, and cell sorting based on Hoechst 33342 perfusion to directly study hypoxia and proliferation in human tumor xenografts and transplantable murine tumors in vivo. Hypoxia was largely confined to cells in regions with the least perfusion, although in tumors exhibiting transient blood flow, hypoxic cells were not as highly localized. Similarly, proliferation and hypoxia were mutually exclusive except in areas of a tumor subjected to transient changes in perfusion. By determining the clonogenic potential, pimonidazole labeling intensity, and radiosensitivity of sorted tumor cell subpopulations, we have provided direct evidence that pimonidazole identifies hypoxic tumor cells of therapeutic relevance in vivo. Given that pimonidazole exhibits few diffusion or delivery problems and no apparent cytotoxicity, it appears to be a versatile and useful label for hypoxic cells in solid tumors.     

Excimer laser-facilitated angioplasty for undilatable coronary narrowings

Calcified and fibrotic coronary artery lesions cannot always be dilated with conventional balloon angioplasty even at high pressures. This study examines the success of excimer laser facilitated angioplasty in 38 lesions in 37 patients with lesions that failed balloon angioplasty alone.     

Cyclosporine in glomerular disease

Cyclosporine was introduced in 1981 as an immunosuppressive agent in renal transplantation. Its use was soon extended to the treatment of various glomerular disorders. In light of its known immunomodulating effects, the use of cyclosporine has been most prominent in those glomerular diseases thought to have an immune basis. The most careful studies of cyclosporine in glomerular diseases have been performed in the pediatric population with idiopathic nephrotic syndrome (i.e., minimal change disease and focal segmental glomerulosclerosis), although data are accumulating regarding efficacy and safety in adults with idiopathic nephrotic syndrome. In patients who are steroid-dependent, cyclosporine therapy can induce complete or partial remission in a significant proportion of cases; success rates in patients with steroid-resistant nephrotic syndrome are less encouraging. Treatment with cyclosporine allows for dose reduction or elimination of corticosteroids, and the consequent salutary effect on growth in the child and glucose and bone metabolism in all patients. Studies that suggest a potential benefit of cyclosporine in recurrent nephrotic syndrome in renal allografts and in other glomerular diseases are also discussed.     

Apoptosis in the failing human heart

BACKGROUND: Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS: Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS: Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS: Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.     

A base-pairing interaction between U2 and U6 small nuclear RNAs occurs in > 150S complexes in HeLa cell extracts: implications for the spliceosome assembly pathway

In mammalian cells, base pairing between the U2 and U6 small nuclear RNAs is required during pre-RNA splicing. We show by psoralen crosslinking of HeLa nuclear extract that U2.U6 base pairing occurs within abundant ribonucleoprotein complexes that sediment at > 150 S in glycerol gradients. All of the spliceosomal RNAs (U1, U2, U4, U5, and U6) cosediment with these large complexes, suggesting that they may be related to small nuclear RNA-containing structures called speckles/coiled bodies or snurposomes, which have been visualized in mammalian or amphibian nuclei, respectively. In contrast to nuclear extract, S100 extract, which is splicing-defective and lacks the > 150S complexes, does not contain base-paired U2.U6. However, U2.U6 base pairs form in S100 extract that has been made splicing-competent by supplementation with Ser/Arg-rich (SR) proteins, ATP, and an adenovirus splicing substrate. During splicing in supplemented S100 extract, U2.U6 base pairing precedes the appearance of splicing intermediates and occurs initially in an approximately 60S spliceosome complex but also in progressively larger (100-300 S) complexes. Possible functional relationships between the 60S spliceosome and the > 150S complexes are discussed.