Fifteenth Aspen Cancer Conference: mechanisms of toxicity, carcinogenesis, and cancer prevention.
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Animals↗
Biomedical subjects
M Sander
Publications and source records attributed to M Sander.
Comparison of static and dynamic disinfection models for bacteria and viruses in water of varying quality.
Disinfection studies rarely use natural waters due to demands exerted on the applied disinfectants and lack of consistent disinfectant residuals. This study compared the degree of disinfection achieved in natural waters between conventional batch (static) models and a system of similar volume where disinfectant residuals were maintained at constant levels (dynamic). In the latter, disinfectant was delivered through a hollow fibre cartridge from a slipstream of a full-scale (chloramine) or pilot (chlorine) water treatment plant. The test organisms (hepatitis A virus, poliovirus, MS-2, Mycobacterium terrae and Enterococcus durans) were selected with different resistance to the disinfectants. In general, for water of "good" quality, the differences between the two systems were often small or not apparent for monochloramine. However, for low chlorine residuals, or when additional demand was placed on the disinfectant, differences between the two systems became more apparent. Little difference was seen between disinfection of the test organisms singly or in mixtures, but injury of vegetative bacteria with monochloramine was very apparent. This system could be useful for understanding the fluctuations in disinfection efficacy that may occur in source water of varying quality, or in distribution systems, as disinfectant residuals decline.
Chlorine↗
Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin-glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to alpha-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD.
Adolescent↗
Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates.
There is growing evidence that sonic hedgehog (Shh) signaling regulates ventral neuronal fate in the vertebrate central nervous system through Nkx-class homeodomain proteins. We have examined the patterns of neurogenesis in mice carrying a targeted mutation in Nkx6.1. These mutants show a dorsal-to-ventral switch in the identity of progenitors and in the fate of postmitotic neurons. At many axial levels there is a complete block in the generation of V2 interneurons and motor neurons and a compensatory ventral expansion in the domain of generation of V1 neurons, demonstrating the essential functions of Nkx6.1 in regional patterning and neuronal fate determination.
Animals↗
Metabolic modulation of sympathetic vasoconstriction in human skeletal muscle: role of tissue hypoxia.
Sympathetically evoked vasoconstriction is modulated by skeletal muscle contraction, but the underlying events are incompletely understood. During contraction, intramuscular oxygenation decreases with increasing exercise intensity. We therefore hypothesized that tissue hypoxia plays a crucial role in the attenuation of sympathetic vasoconstriction in contracting skeletal muscle. In 19 subjects, near-infrared spectroscopy was used to measure decreases in muscle oxygenation (DeltatHbO2+MbO2) as an estimate of the vasoconstrictor response to reflex sympathetic activation with lower body negative pressure (LBNP) in the microcirculation of resting and contracting forearm muscles. Oxygen delivery to the muscles was reduced by decreasing (a) arterial O2 content by breathing 10 % O2, or (b) muscle perfusion by applying forearm positive pressure (FPP, +40 mmHg). In resting forearm, reflex sympathetic activation decreased muscle oxygenation by 11 +/- 1 %. Handgrip alone at 5 and 20 % of maximal voluntary contraction (MVC) decreased muscle oxygenation by 4 +/- 1 and 28 +/- 4 %, respectively. When superimposed on handgrip, LBNP-induced decreases in muscle oxygenation were preserved during handgrip at 5 % MVC, but were abolished during handgrip at 20 % MVC. Oral administration of aspirin (1 g) did not restore the latter response. When the decrease in forearm muscle oxygenation elicited by handgrip at 20 % MVC was mimicked by either (a) systemic hypoxia plus 5 % handgrip (DeltatHbO2+MbO2, -32 +/- 3 %), or (b) hypoperfusion of resting muscle by FPP (DeltatHbO2+MbO2, -26 +/- 6 %), LBNP-induced decreases in muscle oxygenation were greatly attenuated. These data suggest that local tissue hypoxia is involved in the metabolic attenuation of sympathetic vasoconstriction in the microcirculation of exercising human skeletal muscle. The specific underlying mechanism remains to be determined, although products of the cyclo-oxygenase pathway do not appear to be involved.
Adult↗
Great leaps forward: translesion synthesis gets unstalled.
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Adenosine Triphosphatases↗
Metabolic modulation of sympathetic vasoconstriction in exercising skeletal muscle.
The tight coupling of oxygen supply and utilization in exercising skeletal muscle is the result of complex interactions between local mechanisms that control muscle blood flow and substrate utilization and systemic mechanisms that control cardiac output and arterial pressure. The role of the sympathetic nervous system in the integration of these responses, specifically the interaction between sympathetic activation and local vasodilator mechanisms in exercising muscle, has been an active area of research for many years yet remains incompletely understood. The functional consequence of sympathetic activation in exercising skeletal muscle has been the subject of considerable debate. Previous studies in animals and humans have suggested that sympathetic vasoconstricton in active muscle is (a) well maintained and serves to limit active hyperaemia, thereby preventing muscle blood flow from outstripping cardiac output in order to preserve blood pressure and vital organ perfusion or (b) greatly attenuated in order to optimize muscle perfusion, a concept that has been termed 'functional sympatholysis'. Studies performed over the past 70 years have provided conflicting evidence regarding the relative importance of sympathetic vasoconstriction vs. functional sympatholysis in exercising skeletal muscle. The focus of this review is mainly on recent studies in anaesthetized animal preparations and in conscious humans that have provided evidence for the metabolic modulation of sympathetic vasoconstriction in contracting skeletal muscle and have identified a number of key underlying mechanisms that extend the initial concept of sympatholysis.
Animals↗
Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas.
Most insulin-producing beta-cells in the fetal mouse pancreas arise during the secondary transition, a wave of differentiation starting at embryonic day 13. Here, we show that disruption of homeobox gene Nkx6.1 in mice leads to loss of beta-cell precursors and blocks beta-cell neogenesis specifically during the secondary transition. In contrast, islet development in Nkx6. 1/Nkx2.2 double mutant embryos is identical to Nkx2.2 single mutant islet development: beta-cell precursors survive but fail to differentiate into beta-cells throughout development. Together, these experiments reveal two independently controlled pathways for beta-cell differentiation, and place Nkx6.1 downstream of Nkx2.2 in the major pathway of beta-cell differentiation.
Animals↗
Genomic imprinting and environmental disease susceptibility.
Genomic imprinting is one of the most intriguing subtleties of modern genetics. The term "imprinting" refers to parent-of-origin-dependent gene expression. The presence of imprinted genes can cause cells with a full parental complement of functional autosomal genes to specifically express one allele but not the other, resulting in monoallelic expression of the imprinted loci. Genomic imprinting plays a critical role in fetal growth and behavioral development, and it is regulated by DNA methylation and chromatin structure. This paper summarizes the Genomic Imprinting and Environmental Disease Susceptibility Conference held 8-10 October 1998 at Duke University, Durham, North Carolina. The conference focused on the importance of genomic imprinting in determining susceptibility to environmentally induced diseases. Conference topics included rationales for imprinting: parental antagonism and speciation; methods for imprinted gene identification: allelic message display and monochromosomal mouse/human hybrids; properties of the imprinted gene cluster human 11p15.5 and mouse distal 7; the epigenetics of X-chromosome inactivation; variability in imprinting: imprint erasure, non-Mendelian inheritance ratios, and polymorphic imprinting; imprinting and behavior: genetics of bipolar disorder, imprinting in Turner syndrome, and imprinting in brain development and social behavior; and aberrant methylation: methylation and chromatin structure, methylation and estrogen exposure, methylation of tumor-suppressor genes, and cancer susceptibility. Environmental factors are capable of causing epigenetic changes in DNA that can potentially alter imprint gene expression and that can result in genetic diseases including cancer and behavioral disorders. Understanding the contribution of imprinting to the regulation of gene expression will be an important step in evaluating environmental influences on human health and disease.
Animals↗
Neural mechanisms in nitric-oxide-deficient hypertension.
Nitric oxide is hypothesized to be an inhibitory modulator of central sympathetic nervous outflow, and deficient neuronal nitric oxide production to cause sympathetic overactivity, which then contributes to nitric-oxide-deficient hypertension. The biochemical and neuroanatomical basis for this concept revolves around nitric oxide modulation of glutamatergic neurotransmission within brainstem vasomotor centers. The functional consequence of neuronal nitric oxide in blood pressure regulation is, however, marked by an apparent conflict in the literature. On one hand, conscious animal studies using sympathetic blockade suggest a significant role for neuronal nitric oxide deficiency in the development of nitric-oxide-deficient hypertension, and on the other hand, there is evidence against such a role derived from 'knock-out' mice lacking nitric-oxide synthase 1, the major source of neuronal nitric oxide.
Animals↗
A large blood pressure-raising effect of nitric oxide synthase inhibition in humans.
In experimental animals, systemic administration of nitric oxide synthase (NOS) inhibitors causes large increases in blood pressure that are in part sympathetically mediated. The aim of this study was to determine the extent to which these conclusions can be extrapolated to humans. In healthy normotensive humans, we measured blood pressure in response to two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME), the latter of which recently became available for use in humans. The major new findings are 3-fold. First, L-NAME produced robust increases in blood pressure that were more than 2 times larger than those previously reported in humans with L-NMMA and approximated those seen in experimental animals. L-NAME (4 mg/kg) raised mean arterial pressure by 24+/-2 mm Hg (n=27, P<0.001), whereas in subjects who received both inhibitors, a 12-fold higher dose of L-NMMA (50 mg/kg) raised mean arterial pressure by 15+/-2 mm Hg (n=4, P<0.05 vs L-NAME). Second, the L-NAME-induced increases in blood pressure were caused specifically by NOS inhibition because they were reversed by L-arginine (200 mg/kg, n=12) but not D-arginine (200 mg/kg, n=6) and because NG-nitro-D-arginine methyl ester (4 mg/kg, n=5) had no effect on blood pressure. Third, in humans, there is an important sympathetic component to the blood pressure-raising effect of NOS inhibition. alpha-Adrenergic blockade with phentolamine (0.2 mg/kg, n=9) attenuated the L-NAME-induced increase in blood pressure by 40% (P<0.05). From these data, we conclude that pharmacological inhibition of NOS causes large increases in blood pressure that are in part sympathetically mediated in humans as well as experimental animals.
Adult↗
Drosophila Rrp1 domain structure as defined by limited proteolysis and biophysical analyses.
Drosophila Rrp1 is a DNA repair nuclease whose C-terminal region shares extensive homology with Escherichia coli exonuclease III, has nuclease activity, and provides resistance to oxidative and alkylating agents in repair-deficient E. coli strains. The N-terminal 421 amino acid region of Rrp1, which binds and renatures homologous single-stranded DNA, does not share homology with any known protein. Proteolysis by endoproteinase Glu-C (protease V8) reduces the Rrp1 protein to a single, cleavage-resistant peptide. The peptide (referred to as Rrp1-C274) begins with the sequence TKTTV, corresponding to cleavage between Glu-405 and Thr-406 of Rrp1. We determined that nuclease activity is intrinsic to Rrp1-C274 although altered when compared with Rrp1; 3'-exonuclease activity is reduced 210-fold, 3'-phosphodiesterase activity is reduced 6.8-fold, and no difference in apurinic/apyrimidinic endonuclease activity is observed. Rrp1 and Rrp1-C274 are both monomers with frictional coefficients of 2.2 and 1.4, respectively. Circular dichroism results indicate that Rrp1-C274 is predominantly alpha-helical, while the N-terminal 399 amino acids is predominantly random coil. These results suggest that Rrp1 may have a bipartite structural organization; a highly organized, globular C-terminal domain; and an asymmetric, protease-sensitive random coil-enriched N-terminal region. A shape model for this bipartite structure is proposed and discussed.
Amino Acid Sequence↗
Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle.
The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the mdx mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate alpha-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of alpha-adrenergic vasoconstriction in active skeletal muscle.
Animals↗
A novel glucose-responsive element in the human insulin gene functions uniquely in primary cultured islets.
Insulin gene transcription is limited to the beta cells within the mammalian pancreas and, like insulin secretion, is regulated by glucose. Our previous studies in primary cultured beta cells suggested the presence of a strong glucose-responsive enhancer element between base pairs -341 and -260 of the human insulin promoter, the same region in which a transcriptional repressor had been identified in beta-cell tumor lines. In an attempt to map these promoter activities and resolve these conflicting data, we designed minienhancer constructs spanning this region, and tested them in primary cultured and immortalized cells. One sequence, the Z element (base pairs -292 to -243), functions as both a potent glucose-responsive transcriptional enhancer in primary cultured islet cells and as a transcriptional repressor in immortalized beta and nonbeta cells and in primary fibroblasts. In addition, the Z element binds a novel glucose-responsive protein complex that is found in the nuclei of primary cultured islet cells, but not in the nuclei of tumor cells or primary cultured fibroblasts. These data demonstrate a critical role for the Z element in human insulin gene transcription and its regulation by glucose.
Animals↗
Congenital melanocytic nevi with placental infiltration by melanocytes: a benign condition that mimics metastatic melanoma.
BACKGROUND: Placental metastases from cutaneous malignant melanoma from both the mother and the fetus have been reported. The finding of benign-appearing melanocytes in the placenta in association with congenital melanocytic nevi (CMN) is more exceptional, with only 6 reports in the literature. Clinically, the finding of melanocytes in the placenta in this setting can be alarming and might erroneously lead to the diagnosis of metastatic melanoma. OBSERVATIONS: Herein, we describe 3 additional patients with CMN with placental infiltration by melanocytes with a benign phenotype. In the results of immunoperoxidase stains, the melanocytic cells were positive for S-100 protein and HMB-45 in the 2 lesions available for study. Staining of placental vessels with Ulex europaeus agglutinin I (Vector Laboratories, Burlingame, Calif) failed to show intravascular melanocytes in the 1 lesion available for study. We report for the first time DNA diploidy in 2 lesions available for study, which were analyzed by DNA image cytometry. We describe the first patient with a relatively small, nongiant CMN. CONCLUSIONS: We support the notion of the aberrant migration of melanocytes from the neural crest during fetal development as the most likely explanation of this phenomenon and note the similarity to the association of CMN and leptomeningeal melanocytosis. However, the precise histogenesis of this process remains uncertain. Most importantly, our data provide further evidence for the benign nature of this condition. Awareness of this entity is of vital importance in avoiding overdiagnosis of melanoma in this clinical setting.
Adolescent↗
Reactivity of haloketenes and halothioketenes with nucleobases: reactions in vitro with DNA.
Ketenes are important and highly reactive intermediates. Thioketenes are formed by cysteine conjugate beta-lyase-dependent biotransformation of 1-halovinylcysteine S-conjugates which are metabolites of several halogenated olefins. Nucleic acid constituents react with haloketenes and halothioketenes in vitro. Thioketenes induce DNA strand breaks in incubations of 1,2-dichlorovinyl 2-nitrophenyl disulfide, a thioketene precursor, with pBr 322 plasmid DNA. After treatment of single-stranded or native calf thymus DNA with chlorothioketene generated by the hydrolysis of 1,2-dichlorovinyl 2-nitrophenyl disulfide, the formation of 3,N4-thioacetylcytosine could be demonstrated. N6-(Dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine, however, adducts formed by dichloroketene in vitro, are labile to hydrolysis. Therefore, the binding of this compound to DNA constituents in intact DNA is difficult to demonstrate. Substitution of one chlorine atom by fluorine allowed us to use 19F NMR as a tool to demonstrate the formation of adducts by dihaloketenes in intact DNA. N6-(Chlorofluoroacetyl)adenine and N4-(chlorofluoroacetyl)cytosine were synthesized (yields 77%, 15%, respectively) as references and characterized by LC/MS, 1H, 13C, and 19F NMR, FT-IR, and elemental analysis. To demonstrate the ability of dihaloketenes to bind to DNA, poly-dA (1 mg) and calf thymus DNA (10 mg) were suspended in DMF and treated with different concentrations of chlorofluoroketene (50-200 micromol). Analysis of the polymeric DNA by 19F NMR showed one doublet at -137.2 ppm downfield from the reference (CFCl3). A doublet at -146.9 ppm, characteristic for chlorofluoroacetic acid, an expected product of DNA adduct hydrolysis, was not detected. These results demonstrate the formation of a stable adenine adduct by dihaloketenes in intact calf thymus DNA.
Animals↗
Invited editorial on "Effect of arterial occlusion on responses of group III and IV afferents to dynamic exercise".
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Animals↗
Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development.
We present genetic and biochemical evidence that PAX6 is a key regulator of pancreatic islet hormone gene transcription and is required for normal islet development. In embryos homozygous for a mutant allele of the Pax6 gene, Small eye (Sey(Neu)), the numbers of all four types of endocrine cells in the pancreas are decreased significantly, and islet morphology is abnormal. In the remaining islet cells, hormone production, particularly glucagon production, is markedly reduced because of decreased gene transcription. These effects appear to result from a lack of PAX6 protein in the mutant embryos. Biochemical studies identify wild-type PAX6 protein as the transcription factor that binds to a common element in the glucagon, insulin, and somatostatin promoters, and show that PAX6 transactivates the glucagon and insulin promoters.
Alleles↗