Chromatin organisation of transgenes in Dictyostelium.

The introduction of transgenes in Dictyostelium discoideum typically results in the integration of the transformation vector into the genome at one or a few insertion sites as tandem arrays of approximately 100 copies. Exceptions are extrachromosomal vectors, which do not integrate into chromosomes, and vectors containing resistance markers such as blasticidin, which integrate as single copies at one or a few sites. Here we report that low copy number vector inserts display typical euchromatic features while high copy number insertions are enriched for modifications associate with heterochromatin. Interestingly, high copy number insertions also colocalise with heterochromatin, are enriched for the centromeric histone CenH3 and display centromere-like behaviour during mitosis. We also found that the chromatin organisation on extrachromosmal transgenes is different from those integrated into the chromosomes.

The occurrence of actinlike filaments in association with migrating pigment granules in frog retinal pigment epithelium.

In the retina of the frog and certain other animals, melanin pigment granules move in response to light so as to shield photoreceptor outer segments. The granules are contained within the cells of the pigment epithelium (PE) which lie as a continuous sheet between the neural retina and the choroid. Moderate illumination of the eye causes the melanin granules to move from a region within a PE cell body into numerous fingerlike extensions of the cell which interdigitate with the receptor outer segments. This migration takes many minutes and is reversed when the light falling on the eye increases in intensity. Several reviews are concerned with the early descriptions of this phenomenon (6,30) and with more recent experiments (1,5,19). The mechanism of the pigment granule motion is undetermined although there are studies concerning PE ultrastructure (8, 23, 31), scanning electron microscopy of the fingerlike extensions of the PE cells (27), the role of the PE in photoreceptor phagocytosis (32), the nature of the pigment granules (19), and the action spectrum of the light which induces the migration (16). This study reports the presence of a system of microfilaments associated with the pigment granules in the fingerlike extensions processes of the PE cells. We demonstrate by heavy meromyosin (HMM) labeling that the filaments are actinlike in character and suggest that these filaments could be responsible for the migration of the melanin pigment granules.

Abnormal development of kitten retino-geniculate connectivity in the absence of action potentials.

Action potentials were silenced in one eye of neonatal kittens by repeated intraocular injections of tetrodotoxin for 5 to 8 weeks. After tetrodotoxin blockade was allowed to wear off, receptive field properties of individual relay cells in the lateral geniculate nucleus were examined. The many ON-OFF and binocular fields found in the layers that receive input from the treated eye suggest that these cells had extremely abnormal retino-geniculate synaptic connections. These effects were different in kind from those seen after deprivation rearing that does not silence action potentials. Lack of action potential activity was concluded to lead to abnormal development in the central nervous system.

Studies of trypanocidal (inhibitory) power of naphthoquinones: evaluation of quantum chemical molecular descriptors for structure-activity relationships.

Electronic, lipophilic and steric descriptors included in QSAR-2D and -3D are analyzed for a set of ortho- and para-naphthoquinones that have proved to be powerful oxidative agents with potent trypanocidal activities specially against Leptomonas seymouri and Trypanosoma cruzi. Electronic properties are calculated by means of semiempirical (PM3), ab initio (HF/3-21G) and density functional theory (B3LYP/6-31+G*) methodologies. Three different electronic states, neutral quinones, hydroquinones and semiquinones, are studied to investigate if any one of them are statistically related with the biological activities. The best correlations were obtained at the B3LYP level of theory because it includes electronic correlation. The QSAR-2D indicates that the best trypanocidal growth inhibitors are molecules in the semiquinone electronic state, with the following properties: (a) high negative value of EHOMO, (b) high negative charge in the oxygen atoms of the carbonyl groups, (c) high positive charge in the carbon atom of one of carbonyl moieties and (d) high electronegativity (chi). In a complementary way, the QSAR-3D indicates that the electrostatic field correlates with trypanocidal activity and the presence of bulk moieties would increase activity. The idea of comparing the three electronic states may prove to be of most importance in the general strategy to the design of new trypanocidal drugs. In fact, the experimental results showed that semiquinone is the one really statistically relevant indicating a clear connection between biochemical and theoretical aspects. Finally, we demonstrated that to be a good anti-trypanosomatid compound, the molecule must be a good electron acceptor to reach easily the essential semiquinone state. We expect that the present results motivate new experimental as well as theoretical investigations that confirm our findings.

Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay.

Rabbit antiserum prepared against N-(guanosin-8-yl)-acetylaminofluorene was utilized in radioimmunoassay to detect formation and removal of C-8 adducts from the DNA of cultured cells exposed to N-acetoxy-2-acetylaminoflorene. The assay was able to quantitate both acetylated and deacetylated C-8 adducts between 0.5 and 5 pmol while the N2 adduct, 3-(deoxyguanosin-N2-yl)acetylaminofluorene, was not detected below 160 pmol. By varying the proportions of acetylated and deacetylated C-8 adducts in the radioimmunoassay, a series of standard curves were developed from which the relative proportion of each adduct could be determined in unknown mixtures. DNA from mouse epidermal cells and human skin fibroblasts exposed to N-acetoxy-2-acetylaminofluorene in culture contained only 3 and 5% respectively, of the C-8 adduct in the acetylated form. Quantitation by radioimmunoassay of total C-8 adducts bound to DNA yielded values approximately 25% lower than total carcinogen binding determined by radiolabeling. When removal of C-8 adducts was followed over a 23-hr, carcinogen-free culture period, mouse and human cells removed 40 and 50%, respectively, of bound acetylated and deacetylated C-8 adducts. These studies demonstrate the versatility of radioimmunoassay as a molecular probe for studies of chemical carcinogens.

The chemotherapy of chagas' disease: an overview.

The review presents: a) a brief description of the disease; b) a summary of the most important metabolic targets so far identified in Trypanosome cruzi (T. cruzi) along with corresponding inhibitor compounds; c) the current state of knowledge on the trypanothione reductase system of trypanosomatids with reference to oxidative stress defenses; d) detailed discussions on T. cruzi trypanothione reductase inhibitors such as nitrofuranes, naphthoquinones and phenothiazines. As yet, the chemotherapy of Chagas' disease remains an unsolved problem. Further search for new drugs must continue by means of nucleating existing chemotherapy efforts.

Nitrofuran inhibition of microsomal lipid peroxidation.

Two nitrofuran compounds, nifurtimox and nitrofurantoin, inhibited in a concentration-dependent manner the NADPH-, iron-induced lipid peroxidation in rat liver microsomes, as shown by the decreased rate of MDA accumulation. Other nitro compounds (benznidazole and chloramphenicol) were relatively inactive. Nifurtimox inhibition affected polyenoic fatty acids and cytochrome P-450 degradation that follows lipid peroxidation. The ascorbate- or tert-butyl hydroperoxide-dependent lipid peroxidations were much less inhibited than the NADPH-dependent one. Nifurtimox and nitrofurantoin, but not benznidazole and chloramphenicol, strongly stimulated the microsomal NADPH-oxidase activity, thus supporting electron diversion, as the main cause of the inhibition of peroxidation initiation.

Anatomy of the retina of the mink (Mustela vison).

The retina of the normal pigmented mink has been studied by light and electron microscopy. This retina resembles the typical vertebrate retinia in its patterns of lamination and synaptic interconnectivity. Rod and cone outer segments and receptor spherule and pedicle endings are found. At least two different types of horizontal cell processes are seen with the electron microscope, suggestive of rabbit A and B types. Ribbon and conventional synapses are found in both plexiform layers; conventional synapses are also present in the inner nuclear layer. Quantitative studies of the inner plexiform layer revealed amacrine:bipolar synapse ratios (3.3:1) similar to those of the cat and monkey. Other quantitative parameters also resembled those previously reported for species with retinas that predominantly contain concentric-type receptive fields.

Interneuron circuits in the lateral geniculate nucleus of monocularly deprived cats.

This work investigated the function of interneurons and other types of cells in the lateral geniculate nucleus (LGN) in cats raised to adulthood with one eye sutured closed. In order to understand the basis of the commonly found deficit of Y-type relay cells in the deprived layers of the LGN, we looked for reduced or defective activity in other cells which also receive an afferent projection from Y-type ganglion cells in the visually deprived retina. Monocular deprivation did not produce a deficit in the activity of a class of interneurons which receive direct optic inputs from the same ganglion cells in the deprived eye that also drive the Y-type relay cells. Likewise, the Y-type afferent input from the deprived eye to XY-type relay cells was normal. The XY-type cells have mixed or hybrid receptive field properties and both X and Y excitatory inputs; although the Y-inputs to these cells are often much weaker than the X-inputs. The normal properties of Y-type interneurons and XY-type relay cells in the deprived LGN suggest that neither a retinal dysfunction nor an inherent inability of the Y-type optic tract axons to form adequate synapses onto LGN neurons are factors which would readily account for the reduction of Y-type relay cells in monocularly deprived cats. The hypothesis that the deprived Y-type relay cells may have difficulty in forming synaptic connections onto postsynaptic, binocular neurons was supported by observations of responses of cells in the perigeniculate region. Normally, perigeniculate neurons receive a strong binocular input from Y-type relay cells as well as an X-input in at least some cases. In binocular perigeniculate cells of the sutured cats, no inputs from deprived Y-type relay cells could be identified although a longer latency input, typical of that from X-type relay cells, was present.

Postnatal neurogenesis in the kitten retina.

Postnatal neurogenesis in the kitten retina was studied using 3H-thymidine radioautography. Kittens were injected with 3H-thymidine at 1 day, 10 days, 3 weeks or 4 weeks after birth and allowed to survive until 14 weeks of age. Labeled neuronal nuclei were not found in the ganglion cell layer in any of the retinas, but they were seen in the other nuclear layers of the same retinas. In retinas from kittens injected at one day after birth, the peripheral 80% of the length of the retina (in sections cut parallel to the dorsoventral meridian) contained labeled nuclei; the central 20%, around the optic disc, contained no labeled nuclei. Near the ora serrata most nuclei in both inner and outer nuclear layers were labeled. Away from the ora serrata the proportion of labeled to unlabeled nuclei gradually decreased. Labeled nuclei extended farther centrally in the the inner than the outer nuclear layer. The same pattern of labeling was repeated in retinas from kittens injected at ten days after birth, but fewer nuclei were labeled, and the central, unlabeled region around the optic disc was longer--55% of the length of the retina. Only a few nuclei near the ora serrata were labeled in retinas from kittens injected at three weeks after birth, and no labeled neurons were found in kittens injected at four weeks. From these results we conclude that all of the ganglion cells in the kitten retina are present by one day after birth, as are all of the other neurons in the central retina. In peripheral regions of the inner and outer nuclear layers, proliferation of cells destined to become neurons continues up to three weeks after birth.

Non-uniform postnatal growth of the cat retina.

The distributions of alpha-type ganglion cells in 3-week-old and adult cats were used to measure the increase in the distances between existing cells and thus the amount of growth in various regions of the retina. Growth shows two major non-uniformities. (1) The area centralis is at the point of minimum growth; its area increases by only about 3% while regions near the retinal margin increase in area by about 80%. (2) The retina grows about half as much in linear extent as does the radius of the eye and thus comes to occupy a smaller fraction of the globe. Measurements of retinal dimensions indicate that both non-uniformities also occur from birth to 3 weeks. These non-uniformities have the following implications. (1) They would tend to elongate dendritic fields radially, in the direction of the area centralis, in central retina but perpendicular to this direction in peripheral retina. However, these asymmetries are probably not the primary reason why ganglion cells throughout the retina tend to have radially oriented dendritic fields (Leventhal and Schall, '83). (2) Greater growth in the periphery could contribute to the gradient of increasing dendritic field size from central to peripheral retina if the dendritic fields of ganglion cells passively stretched as the retina expanded. Passive stretching is not the primary determinant of dendritic extent, however, because the dendritic fields of beta-type ganglion cells were found to grow 70% more from 3 weeks to adulthood than can be accounted for by passive stretching. (3) Greater peripheral growth steepens the central-to-peripheral gradient of decreasing ganglion cell density; if this trend also occurs prenatally, it could be the major factor in producing the final adult gradient.

Kitten ganglion cells: dendritic field size at 3 weeks of age and correlation with receptive field size.

A Golgi study of beta (brisk-X type) ganglion cells has been done to compare ganglion cells in the retina of 3-week-old and adult cats. An anatomical basis for the large receptive field centers found in the immature kitten retina was sought. Kitten beta-type ganglion cells have significantly smaller dendritic spreads than adult beta cells; the dendrites of the kitten cells must still grow to reach their final adult size. Therefore a synaptic basis for the large receptive field size of the immature cells is suggested.

Effect of nitroheterocyclic drugs on lipid peroxidation and glutathione content in rat liver extracts.

Incubation of rat liver cell-free extracts with an NADPH-generating system and with nifurtimox or benznidazole (two nitroheterocyclic drugs used in the treatment of Chagas' disease) produced oxidation of reduced glutathione (GSH) and increased lipid peroxidation, as shown by the generation of thiobarbituric-acid-reacting intermediates. Nifurtimox and benznidazole inhibited GSSG-reductase, but not GSH-peroxidase, the former inhibition contributing to GSH depletion. In every case, nifurtimox was more effective than benznidazole. Addition of GSH or free-radical scavengers (catalase, superoxide dismutase, mannitol, sodium benzoate or L-histidine) prevented the effect of nifurtimox on lipid peroxidation reactions. These results support the assumption [M. Dubin, S. N. J. Moreno, E. E. Martino, R. Docampo and A. O. M. Dubin, Biochem. Pharmac. 32, 483 (1983)] that, in the rat liver, GSH exerts a protective action against oxygen radicals generated by the nitroheterocyclic drugs.

Effects of mansonones on lipid peroxidation, P450 monooxygenase activity, and superoxide anion generation by rat liver microsomes.

Several structurally related ortho-naphthoquinones isolated from Mansonia altissima Chev (mansonones C, E and F) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P450 destruction; (c) inhibited NADPH-supported aniline 4-hydroxylase activity; (d) inhibited Fe(III)ADP reduction by NADPH-supplemented microsomes; (e) stimulated superoxide anion generation by NADPH-supplemented microsomes; and (f) stimulated ascorbate oxidation. ESR investigation of ascorbate-reduced mansonone F demonstrated semiquinone formation. Mansonone C had a greater effect than mansonones E and F on NADPH-dependent lipid peroxidation, O2- production and ascorbate oxidation, whereas mansonone E was more effective than mansonones C and F on aniline 4-hydroxylase activity. Mansonones E and F did not inhibit hydroperoxide-dependent lipid peroxidation, cytochrome P450 destruction or microsomal aniline 4-hydroxylase activity. Mansonone C inhibited to a limited degree tert-butyl hydroperoxide-dependent lipid peroxidation, this inhibition being increased by NADPH. Mansonone A, a tetrahydro orthonapthoquinone derivative, was in all respects relatively less effective than mansonones C, E and F. It is postulated that mansonones C, E and F inhibited microsomal lipid peroxidation and cytochrome P450 catalyzed reactions by diverting reducing equivalents from NADPH to dioxygen, but mansonone C (including its reduced form) may also exert direct antioxidant activity.

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by beta-lapachone and related naphthoquinones.

The lipophilic o-naphthoquinones beta-lapachone, 3,4-dihydro-2-methyl-2-ethyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 8-935), 3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 9-442), and 3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione (CG 10-248) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P-450 destruction; (c) inhibited microsomal aniline 4-hydroxylase, aminopyrine N-demethylase and 7-ethoxycoumarin deethylase; (d) did not inhibit the ascorbate- and tert-butyl hydroperoxide-dependent lipid peroxidation and the cumenyl hydroperoxide-linked aniline 4-hydroxylase reaction; and (e) stimulated NADPH oxidation, superoxide anion radical generation and Fe(III)ADP reduction by NADPH-supplemented microsomes. In the presence of ascorbate, the same o-naphthoquinones stimulated oxygen uptake and semiquinone formation, as detected by ESR measurements. The p-naphthoquinones alpha-lapachone and menadione were relatively less effective than the o-naphthoquinones. These observations support the hypothesis that, in the micromolar concentration range, o-naphthoquinones inhibit microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions, by diverting reducing equivalents from NADPH to dioxygen.

Effect of 5-nitroindole on adenylate energy charge, oxidative phosphorylation, and lipid peroxidation in rat hepatocytes.

5-Nitroindole (NI), a mutagenic nitroarene, was assayed for cytotoxic effects on rat hepatocytes. After incubation with 25-100 microM NI, the adenylate energy charge of the hepatocytes decreased significantly as a result of the decrease in ATP and the increase in AMP. ATP depletion correlated well with the effects of NI on mitochondrial electron transfer and energy transduction in hepatocytes. Thus, NI (a) inhibited the antimycin-sensitive hepatocyte respiration; (b) inhibited NADH oxidation by disrupted hepatocyte mitochondria; (c) inhibited L-malate-L-glutamate oxidation by ADP-supplemented mitochondria; (d) in the absence of ADP, stimulated the same substrates and also succinate oxidation by mitochondria; (e) released the latent ATPase activity of mitochondrial F1F0-ATP synthase; (f) shifted the redox level of reduced cytochromes (c + c1) and b towards the oxidized state; (g) inhibited NADH oxidation by disrupted mitochondria in the vicinity of the NADH-dehydrogenase flavoprotein; (h) inhibited Ca2+ uptake by mitochondria using L-malate-L-glutamate as an energy source; (i) inhibited valinomycin-induced, endogenously energized K+ uptake, with little effect on the ATP-induced uptake; and (j) inhibited the MgATP-dependent contraction of Ca(2+)-swollen mitochondria. NI inhibited lipid peroxidation in hepatocytes and also in substrate-supplemented liver microsomes and mitochondria, thus ruling out hydroperoxides as a cause of NI cytotoxicity. Long-term incubation with NI produced loss of hepatocyte viability, as indicated by lactate dehydrogenase leakage.

Increased biliary secretion and loss of hepatic glutathione in rat liver after nifurtimox treatment.

Treatment of rats with nifurtimox, a nitrofuran derivative widely used for the treatment of Chagas' disease, induced a time- and dose-dependent depletion of liver glutathione, maximal effects being obtained with 200 mg nifurtimox/kg body weight. Extra release of both oxidized (GSSG) and reduced (GSH) glutathione into bile contributed to this depletion. Glutathione excretion into bile accounted for only part of liver glutathione loss, thus indicating that, in addition to the GSH-peroxidase reaction (resulting in GSSG generation), other glutathione-related processes were involved in nifurtimox detoxification. Bile flow, bile salt excretion, liver lipid conjugated diene content, liver glutathione reductase and glutathione peroxidase activities, and serum alanine aminotransferase (ALAT) activity were not affected by the nifurtimox treatment, thus ruling out widespread damage of the liver cell by nifurtimox. Nevertheless, the extra GSH release in the nifurtimox-treated rats may indicate an alteration of the hepatocyte membrane.

Cytochemical polarity in lateral geniculate interneurons.

To determine the cytochemical composition of presynaptic dendrites, we have examined the distribution of synapsin 1, calcium and calmodulin-dependent protein kinase II (CaM-II), microtubule-associated protein 2 (MAP-2) and spectrin in cat lateral geniculate (LGN) class III cells by immune-EM. Special attention was paid to the dendrites of these interneurons because they are both pre- and postsynaptic. The dendritic proteins MAP-2 and RBC spectrin were not observed in interneuron dendrites but these proteins were localized in relay cell dendrites. The synaptic vesicle-associated protein synapsin 1 was present in all synaptic vesicle containing profiles, including dendritic terminals. CaM-II, the major postsynaptic density protein, was found in all dendrites. Thus, the LGN interneuron dendritic compartment displays both axonal and dendritic cytochemical properties. The results suggest the possibility of unique molecular interactions in interneuron dendritic terminals.

Prior activity influences the velocity of impulses in frog and cat optic nerve fibers.

Under normal visual stimulation, simultaneous recording from ganglion cells in the retina and from the axons of these cells in the brain revealed activity-dependent differences in the velocity of impulse propagation. In frog ganglion cells, spikes initiated 5-500 ms after a previous impulse showed supernormal increases in conduction velocity of up to 17%; spikes initiated 500-2000 ms after a previous one traveled more slowly than a spike initiated after a long period of rest. Cat ganglion cell impulses showed much smaller supernormality (maximum 3%), but exhibited pronounced slowing due to refractoriness and long-term fatigue associated with their high levels of spontaneous activity.

Response properties of neurons in the lateral geniculate nucleus of neonatal kittens.

A previous study led us to consider the implications of the types of receptive fields found in the lateral geniculate nucleus (LGN) of neonatal kittens. Thus, we studied cells in the A layers in the LGN of kittens aged 6-29 days using extracellular recording techniques. Peri-stimulus-time-histograms were constructed in response to flashing spots of light centered in the receptive field of each unit. All units studied showed an excitatory response only to light onset (on-center) or light offset (off-center). No units were found which had an excitatory response to both phases of the stimulus (On-Off). Possible differences in classification between this study and that of earlier workers who reported On-Off cells in young kittens are discussed.