Pyridine Nucleotide Changes In Hepatocytes Exposed To Quinones

Quinones may be toxic by a number of mechanisms. including arylation and oxidative stress caused by redox cycling. Using isolated hepatocytes, we have studied the cytotoxicity of four quinones. with differing abilities to arylate cellular nucleophiles and redox cycle. in relation to their effects on cellular pyridine nucleotides. High concentrations of menadione (redox cycles and arylates). 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) 2.3-dimethoxy-1.4-naphthoquinone (a pure redox cycler) and p-benzoquinone (a pure arylator) caused an initial decrease in NAD⁺ and loss of viability, which was not prevented by 3-aminobenzamide. an inhibitor of poly(ADP-ribose)polymerase. In contrast. 3-aminobenzamide inhibited the loss of NAD' and viability caused by dimethyl sulphate so implicating poly(ADP-ribose)polymerase in its toxicity but not that of the quinones. Non-toxic concentrations of menadione. 2.3-dimethoxy-1.4-naphthoquinone and 2-hydroxy-1.4-naphthoquinone all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD⁺ was accompanied by a small. transient increase in NADP⁺ and followed by a larger. prolonged increase in NADPH and total NADP⁺ + NADPH. Nucleotide changes were not observed with non-toxic concentrations of p-benzoquinone. Our findings suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides. in an attempt to combat the effects of oxidative stress     

Identification of consensus patterns in unaligned DNA sequences known to be functionally related

We have developed a method for identifying consensus patternsin a set of unaligned DNA sequences known to bind a common proteinor to have some other common biochemical function. The methodis based on a tnatrix representation of binding site patterns.Each row of the matrix represents one of the four possible bases,each column represents one of the positions of the binding siteand each element is determined by the frequency the indicatedbase occurs at the indicated position. The goal of the methodis to find the most significant matrix-i.e. the one with thelowest probability of occurring by chance-out of all the matricesthat can be formed from the set of related sequences. The reliabilityof the method improves with the number of sequences, while thetime required increases only linearly with the number of sequences.To test this method, we analysed 11 DNA sequences containingpromoters regulated by the Escherichia coli LexA protein. Thematrices we' found were consistent with the known consensussequence, and could distinguish the generally accepted LexAbinding sites from other DNA sequences. Received on November 6, 1989; accepted on December 20, 1989     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Diurnal variation of corticotropin-releasing factor binding sites in the rat brain and pituitary

Summary 1. Corticotropin-releasing factor (CRF) is thought to be involved in the regulation of the diurnal activity of the hypothalamus-pituitary-adrenal (HPA) axis and to act as a neurotransmitter in the brain. To date it is unknown whether the binding sites of the central CRF system are subject to diurnal variations. 2. We measured the number of CRF binding sites over the course of a complete 24-hr light-dark cycle in the pituitary, amygdala, bed nucleus of the stria terminalis (BNST), cingulate cortex, visceral cortex, paraventricular nucleus of the hypothalamus, hippocampus, and locus ceruleus of rats byin vitro receptor autoradiography with iodinated ovine CRF. A 24-hr time course was also established for plasma CRF and corticosterone. 3. The diurnal pattern of plasma CRF does not correlate with the pattern of plasma corticosterone. Within the brain, CRF binding in the basolateral nucleus of the amygdala showed a U-shaped curve with maximum levels in the morning and a wide hallow between 1500 and 0100. A biphasic profile with a small depression in the afternoon and a more pronounced depression in the second half of the activity period is characteristic for the other brain areas and the pituitary. The profile for the pituitary correlates with those for the BNST and the area of the locus ceruleus. Furthermore, the diurnal pattern of CRF binding sites in the BNST correlates with that of the hippocampus, and the daytime pattern of the visceral cortex is similar to that of both the hippocampus and the BNST. 4. Since the CRF-binding profiles in the brain and the pituitary clearly differ from the profiles of both plasma CRF and corticosterone, one may assume that the diurnal pattern of central CRF binding sites is not directly coupled to the activity of the HPA axis.     

Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (Se^o). A laboratory strain ofBacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na₂SeO₃). After harvest, the washed, lyophilizedB. subtilis andM. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of Se^o. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray Se^o had a bioavailability of 2% of selenite, but that the bioavailability of red Se^o depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray Se^o and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3–4%). These findings suggest that aerobic bacteria such asB. subtilis andM. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.     

Polysaccharide concentration and molecular weight effects on the oxygen mass transfer in a reciprocating plate bioreactor

In most polysaccharide fermentations, the nature of the fermentation broth changes drastically with time and, as a result, the overall oxygen mass transfer coefficient (K(L)a) can vary by orders of magnitude. To obtain a better understanding of this phenomenon, an experimental program was devised to study the respective influence of molecular weight and concentration of dextran solutions on K(L)a. Experiments were conducted in a reciprocating plate bioreactor. This bioreactor uses a stack of perforated plates that is reciprocated axially in the column and it is therefore well suited for mixing viscous liquid broths and providing uniform overall mass transfer coefficients. The variation of K(L)a with the power input per unit volume and the superficial gas velocity were obtained for three ranges of molecular weights and five concentrations of dextran. In every medium, two regimes of operation were observed as a function of the power input per unit volume: a first regime, at low power inputs per unit volume where K(L)a remains constant until a threshold of power input is attained; and a second regime, which is characterized by a steep increase of K(L)a as a function of the power input per unit volume. The presence of dissolved biological macromolecules, not only because of their effect on the rheology of the medium but also because their effect on the gas-liquid interface, has a significant impact on K(L)a. It was found that, generally, small concentrations of polysaccharide favor oxygen mass transfer despite the increase in medium viscosity. However, the respective influence of polysaccharide concentration and molecular weight was different for the two regimes of operation. (c) 1996 John Wiley & Sons, Inc.     

Fate of the anterior neural ridge and the morphogenesis of the xenopus forebrain

The fate of the anterior neural ridge was studied by following the relative movements of simultaneous spot applications of DiI and DiO from stage 15 through stage 45. These dye movements were mapped onto the neuroepithelium of the developing brain whose shape was gleaned from whole-mount in situs to neural cell adhesion molecule and dissections of the developing nervous system. The result is a model of the cell movements that drive the morphogenesis of the forebrain. The midanterior ridge moves inside and drops down along the most anterior wall of the neural tube. It then pushes forward a bit, rotates ventrally during forebrain flexing, and gives rise to the chiasmatic ridge and anterior hypothalamus. The midanterior plate drops, forming the floor of the forebrain ventricle, and, keeping its place behind the ridge, it gives rise to the posterior hypothalamus or infundibulum. The midlateral anterior ridge slides into the lateral anterior wall of the neural tube and stretches laterally into the optic stalk and retina, and then rotates into a ventral position. The lateral anterior ridge converges to the most anterior part of the dorsal midline during neural tube closure, then rotates anteriorly, and gives rise to telencephalic structures. Whole-mount bromodeoxyuridine labeling at these stages showed that cell division is widespread and relatively uniform throughout the brain during the late neurula and early tailbud stages, but that during late tailbud stages cell division becomes restricted to specific proliferative zones. We conclude that the early morphogenesis of the brain is carried out largely by choreographed cell movements and that later morphogenesis depends on spatially restricted patterns of cell division. © 1995 John Wiley & Sons, Inc.     

Age-related differences in TGF-α and proto-oncogenes expression in rat liver after a low dose of carbon tetrachloride

The resiliency of rats during early postnatal development to CCl₄ or to an interactive hepatotoxicity of chlordecone (CD) + CCl₄ has been shown to be due to an efficient stimulation of tissue repair. The objective of the current study was to investigate if this is due to efficient expression of transforming growth factor-α (TGF-α) and proto-oncogenes. Postnatally developing (20 day old) and adult (60 day old) male Sprague–Dawley rats were challenged with a single low dose of CCl₄ (100 μL/kg, ip) or corn oil. Liver samples were collected during a time course (0–96 h) after the administration of CCl₄ and used to examine TGF-α and early (c-fos) and late (H-ras and K-ras) proto-oncogenes mRNA expressions. Significant increases in TGF-α, H-ras, and K-ras gene expressions were evident as early as 12 hours after CCl₄ and peaked between 24 and 48 hours in an age-dependent manner as detected by slot-blot analysis. Results of the study revealed three- and twofold increases in TGF-α gene expression in 20 and 60 day old rats, respectively, after CCl₄. There were 3.5- and 2.5-fold increases in H-ras and 4.4- and 3.4-fold increases in K-ras in 20 and 60 day old rats, respectively. In contrast, a 10-fold increase in c-fos mRNA expression was evident in 20 day old rats 1 hour after CCl₄ treatment, returning to the baseline value by 3 hours, whereas in 60 day old rats, this increase was less than twofold. The overall findings of this study indicate that TGF-α and the early and late proto-oncogene mRNA expressions were enhanced in an age- and time-dependent manner in response to a low dose of CCl₄. These results further strengthen the view that the remarkable resiliency of rats to hepatotoxicants during early postnatal development is due to substantial increases in stimulation of hepatocellular regeneration and tissue repair mechanisms, leading to regression of liver injury and recovery. © 1996 John Wiley & Sons, Inc.     

Cytogenetic analysis of chromosome 5 from the Mediterranean fruit fly,Ceratitis capitata

A cytogenetic map of chromosome 5 from the Mediterranean fruit fly, Ceratitis capitata, was constructed. Six mutations were located by translocation, transposition or deletion mapping. This knowledge allowed alignment and orientation of the existing linkage map with the polytene chromosomes. In addition, mapping of mutations used as selectable markers in genetic sex-separation strains is an essential prerequisite for the improvement of genetic stability during mass-rearing.     

1,1-Organoboration of tri-1-alkynyltin compounds: novel triorganotin cations, stannoles, 3-stannolenes and 1-stanna-4-bora-2,5-cyclohexadienes

Tri-1-alkynyltin compounds [R²Sn(CCR¹)₃ (1), R² = Me, R¹ = Me (a), ⁿBu (b), ^tBu (c), Me₃Si (d), 1-(1-cyclohexenyl) (e); R² = Et, R¹ = Me (a(Et)), ⁿBu (b(Et)), ^tBu (c(Et)), SiMe₃ (d(Et)); R² = ⁿBu, R¹ = Me (a(Bu)), ⁿBu (b(Bu))] were prepared, and their reactivity towards trialkylboranes Et₃B (2) and ⁱPr₃B (3) in 1,1-organoboration reactions was studied. The first step in each reaction is an intermolecular 1,1-alkytoboration. Afterwards, intramolecular 1,1-vinyloboration or 1,1-alkyloboration compete with further intermolecular 1,1-alkyloboration. Various triorganotin cations (4-7), stabilized by intramolecular side-on coordination to the CC bond of an alkynylborate moiety, were detected as highly fluxional intermediates prior to rearrangement into heterocyclic systems such as stannoles (9-11), 1-stanna-4-bora-2, 5-cyclohexadienes (8, 12). The reactions between 1a or 1a(Bu) and an excess of Et₃B (2) afford the tris(alkenyl)tin compounds 13 via threefold intermolecular 1, 1-ethyloboration. 13 rearrange to the 3-stannolenes (14a or 14a(Bu)). The intermediates and final products were characterized by multinuclear one- and two-dimensional ¹H, ¹¹B, ¹³C, ²⁹Si and ¹¹⁹Sn NMR.