Aromatic amine DNA adduct formation in chronically-exposed mice: considerations for human comparison

Lifetime chronic exposure of mice to the aromatic amines 4-aminobiphenyl (ABP) and 2-acetylaminofluorene (AAF) produces liver and urinary bladder tumors. In parallel experiments, DNA adduct levels in target tissues reach a steady-state (a balance between adduct formation and removal) after about four weeks of either AAF or ABP ingestion. For these and other carcinogens, steady-state DNA adduct levels most frequently increase linearly with dose, but the formation of tumors also depends upon a variety of factors, including the proliferative capacity of the target tissue, the sex of the animal, genotoxic properties of the specific adducts formed, and other unknown events. Chronic dosing experiments in animal models are of interest for human risk assessment because human exposure is typically intermittent, involving repeated exposures. However, it is to be expected that in a genetically-diverse human population, where the lifetime averages > 70 years, the relationship between tumorigenesis and DNA adduct formation will be relatively more complex than that observed in mice. From our studies of chronic ABP exposure in male mice, we have obtained the daily dose of ABP and the steady-state level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct associated with a 50% mouse bladder tumor incidence. Our attempt at a human extrapolation for adducts and urinary bladder cancer in smoking males (20-40 cigarettes/day) is based on the ABP dose per cigarette, values for the dG-C8-ABP adduct in bladder biopsies of lifetime heavy smokers at age approximately 70, and the smoking-related bladder tumor incidence (absolute lifetime risk) for Caucasian males in the United States aged 65-84 years. The extrapolation has produced two major predictions, one related to adduct formation and the other related to tumorigenesis. First, the observed level of smoking-related dG-C8-ABP in DNA of human bladder epithelium, expressed as a function of daily ABP intake, is about 3500-times higher than similar data for mice, which suggests that humans may perform the biotransformation of ABP more efficiently than mice. Second, at a similar bladder tumor incidence, mouse bladder contained adduct concentrations that were much higher than those observed in human bladder; for example, at a 2.6% tumor incidence, mouse bladder contained an average of 55.5 fmol dG-C8-ABP/microgram DNA (1850 adducts/10(8) nucleotides), while bladders from Caucasian male smokers contained an average of 0.036 fmol dG-C8-ABP/microgram DNA (1.2 adducts/10(8) nucleotides). This suggests that factors other than ABP-DNA adducts, such as adducts of other carcinogens, the influence of promoters, and synergistic effects of all of these factors contribute substantially to smoking-related bladder cancer in humans.     

Status epilepticus-induced alterations in metabotropic glutamate receptor expression in young and adult rats

In adult rats, kainic acid induces status epilepticus and delayed, selective cell loss of pyramidal neurons in the hippocampal CA3. In pup rats, kainate induces status epilepticus but not the accompanying neuronal cell death. The precise mechanisms underlying this age-dependent vulnerability to seizure-induced cell death are not understood. Metabotropic glutamate receptors (mGluRs) are developmentally and spatially regulated throughout the hippocampus and are implicated in seizure-induced damage. In the present study we used in situ hybridization to examine possible changes in mGluR expression at the level of the hippocampus after status epilepticus in postnatal day 10 (P10) pup and adult (P40) rats. Status epilepticus did not alter expression of mGluR1, mGluR3, or mGluR5 mRNAs. In pup and adult rats, status epilepticus induced a reduction in expression of mGluR2 mRNA in granule cells of the dentate gyrus. This change could lead to augmented glutamate release at mossy fiber synapses on CA3 pyramidal cells and thereby promote hyperexcitation. In pup but not adult rats, mGluR4 mRNA expression was enhanced in CA3 pyramidal neurons. Upregulation of presynaptic mGluR4 in pup CA3 neurons could lead to reduced transmitter release from CA3 axons, including recurrent collaterals, thereby reducing vulnerability of neonatal CA3 neurons to seizure-induced damage. These findings indicate that status epilepticus affects mGluR expression in a gene- and cell-specific manner, and that these changes vary with the developmental stage.     

Sequence specificity and biochemical characterization of the RusA Holliday junction resolvase of Escherichia coli

The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5' of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses approximately 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+ strains unless it is expressed at a high level.     

Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced beta2 integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha (TNF-alpha), and this effect was also mediated by beta2 integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by beta2 integrins with a decrease in the fraction of PMN rolling on TNF-alpha-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced beta2 integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.     

Systematic screening for fragile X syndrome in a cohort of 574 mentally retarded children

The Sense of Coherence Scale is used frequently world-wide and is thought to be appropriate to any population. The purpose of this study was to highlight some of the potential limitations of the scale. Fifteen members of the Pentecostal Movement described several difficulties in completing the scale, especially items measuring manageability and comprehensibility. The following three statements indicate the nature of their interpretation of the items and the ambiguity of their answers as they argued that: (1) the concept of 'I' was interpreted as 'I and God', (2) it is not necessary to understand everything in life, because it is enough that God understands, and (3) life is meaningful in itself because of the salvation. According to this interpretation of the items, every proposed answer could be suitable and consequently, the scores can readily be misconstrued. Many of the respondents proposed additions or changes in the wording to make the scale more suitable. The strong ego-centred items seem to be inappropriate for the participants in this study and other populations might also be confronted with similar difficulties.     

Choline kinase inhibitors as a novel approach for antiproliferative drug design

Recent progress in deciphering the molecular basis of carcinogenesis is of utmost importance to the development of new anticancer strategies. To this end, it is essential to understand the regulation of both normal cell proliferation and its alterations in cancer cells. We have previously demonstrated that in ras-transformed cells there is an increased level of phosphorylcholine (PCho) resulting from a constitutive activation on choiline kinase (ChoK). The importance of ChoK for the regulation of cell proliferation has also been proposed since an inhibitor for this enzyme, hemicholinium-3 (HC-3), drastically reduces entry into the S phase after stimulation with growth factors. Here we report the synthesis of several new compounds which are highly specific inhibitors for ChoK, with up to 1000-fold or 600-fold increased inhibitory activity, compared to HC-3 under ex vivo or in vitro conditions respectively. These novel compounds also drastically reduce entry into the S phase after stimulation with specific growth factors. A more profound inhibition of cell proliferation was observed in ras-, src- and mos-transformed cells in the presence of ChoK inhibitors, compared to their parental, untransformed NIH3T3 cells. By contrast, this effect was not observed in fos-transformed cells. While ras, src and mos transformation is associated with elevated levels of ChoK activity, fos-induced transformation does not affect ChoK activity. The inhibitory effect on proliferation of the new compounds correlates with their ability to inhibit the production of phosphorylcholine in whole cells, a proposed novel second messenger for cell proliferation. These results strongly support a critical role of choline kinase in the regulation of cell growth and makes this enzyme a novel target for the design of new antiproliferative and anticancer drugs.     

Evaluation of mental retardation: recommendations of a Consensus Conference: American College of Medical Genetics

A Consensus Conference utilizing available literature and expert opinion sponsored by the American College of Medical Genetics in October 1995 evaluated the rational approach to the individual with mental retardation. Although no uniform protocol replaces individual clinician judgement, the consensus recommendations were as follows: 1. The individual with mental retardation, the family, and medical care providers benefit from a focused clinical and laboratory evaluation aimed at establishing causation and in providing counseling, prognosis, recurrence risks, and guidelines for management. 2. Essential elements of the evaluation include a three-generation pedigree: pre-, peri-, and post-natal history, complete physical examination focused on the presence of minor anomalies, neurologic examination, and assessment of the behavioral phenotype. 3. Selective laboratory testing should, in most patients, include a banded karyotype. Fragile X testing should be strongly considered in both males and females with unexplained mental retardation, especially in the presence of a positive family history, a consistent physical and behavioral phenotype and absence of major structural abnormalities. Metabolic testing should be initialed in the presence of suggestive clinical and physical findings. Neuroimaging should be considered in patients without a known diagnosis especially in the presence of neurologic symptoms, cranial contour abnormalities, microcephaly, or macrocephaly. In most situations MRI is the testing modality of choice. 4. Sequential evaluation of the patient, occasionally over several years, is often necessary for diagnosis, allowing for delineation of the physical and behavioral phenotype, a logical approach to ancillary testing and appropriate prognostic and reproductive counseling.     

Oculocerebral dysgenesis in the linear nevus sebaceous syndrome

PURPOSE: The authors document the association of peripapillary staphyloma and an atypical variant of hemimegalencephaly with the linear nevus sebaceous syndrome. BACKGROUND: Linear nevus sebaceous syndrome is an uncommon neurocutaneous disorder that has a propensity to involve the eyes. METHODS: Clinical, histopathologic, and neuroimaging findings are examined in a child with linear nevus sebaceous syndrome. RESULTS: In addition to bilateral peripapillary staphylomas, ophthalmologic abnormalities included a corneal dermoid, a complex conjunctival choristoma, macular hypoplasia, and optic nerve hypoplasia with contralateral optic atrophy. Magnetic resonance imaging disclosed a rare form of hemimegalencephaly characterized by hypoplasia of an optic radiation within the enlarged, dysgenetic cerebral hemisphere. CONCLUSION: The spectrum of oculocerebral dysgenesis in the linear nevus sebaceous syndrome can be expanded to include peripapillary staphyloma and atypical hemimegalencephaly with hypoplasia of an optic radiation.     

Further studies on the immunopathology of atopic keratoconjunctivitis using flow cytometry

Atopic keratoconjunctivitis (AKC) is an ocular manifestation of systemic hypersensitivity. Although the pathogenesis of AKC is not fully understood, some previous data suggest that a decrease in numbers of suppressor T lymphocyte (Ts) and increase of Th, especially Th2 (the second subgroup of helper T lymphocyte), at the ocular surface may play an important role in the occurrence of the disease. In this study, the percentages of naive-Th (CD4/45RA+) and memory-Th (CD4/29+) cells, and the Th/Ts and memory-Th cells/naive-Th cells ratios were measured in the blood and tear samples of patients with AKC, atopic patients without ocular involvement and normal volunteers, using flow cytometry. Groups were compared using the Mann-Whitney U test. We found that patients with AKC had significantly higher memory-Th cell concentration, and Th/Ts and memory-Th cells/naive-Th cell ratios both in the tear and blood samples compared to normal subjects. While no significant difference existed between the tear samples of the atopic patients without ocular involvement and normal volunteers with respect to the above values, atopic patients had higher percentages of memory-Th cells and higher Th/Ts and memory-Th cells/naive-Th ratios in their blood than normal subjects. The percentages of memory-Th cells, and the Th/Ts and memory-Th cells/naive-Th cell ratios in the tear samples of AKC patients were also found to be higher than that of the atopic patients without ocular involvement, but no significant difference was present between the blood samples of these groups. The percentages of naive-Th cells did not show any significant difference between groups either in tear or blood samples. Since the mean memory-Th cells/naive-Th1 cells ratio in the tear samples of the patients with AKC was higher than in their blood samples, we propose that the localized accumulation of memory-Th2 cells, in addition to the increase of Th/Ts ratios in the external eye may cause AKC in atopic individuals.     

Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog

Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein alpha subunit homologs in yeast, regulates pseudohyphal differentiation. Deltagpa2/Deltagpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2(Gly19Val) mutant or exogenous cAMP suppresses the Deltagpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels.