Chloroquinoxaline sulfonamide (NSC 339004) is a topoisomerase IIalpha/beta poison.

Chloroquinoxaline sulfonamide (chlorosulfaquinoxaline, CQS, NSC 339004) is active against murine and human solid tumors. On the basis of its structural similarity to the topoisomerase IIbeta-specific drug XK469, CQS was tested and found to be both a topoisomerase-IIalpha and a topoisomerase-IIbeta poison. Topoisomerase II poisoning by CQS is essentially undetectable in assays using the common protein denaturant SDS, but easily detectable with strong chaotropic protein denaturants. The finding that detection of topoisomerase poisoning can be so dependent on the protein denaturant used in the assay has implications for drug discovery efforts and for our understanding of topoisomerase poisons.

Extreme weather and air pollution effects on cardiovascular and respiratory hospital admissions in Cyprus.

In many regions of the world, climatic change is associated with increased extreme temperatures, which can have severe effects on mortality and morbidity. In this study, we examine the effect of extreme weather on hospital admissions in Cyprus, for inland and coastal areas, through the use of synoptic weather classifications (air mass types). In addition, the effect of particulate air pollution (PM10) on morbidity is examined. Our results show that two air mass types, namely (a) warm, rainy days with increased levels of water vapour in the atmosphere and (b) cold, cloudy days with increased levels of precipitation, were associated with increased morbidity in the form of hospital admissions. This was true both for cardiovascular and respiratory conditions, for all age groups, but particularly for the elderly, aged over 65. Particulate air pollution was also associated with increased morbidity in Cyprus, where the effect was more pronounced for cardiovascular diseases.

Quantitative analysis of carbodiimide modified DNA and immunoprobing by adduct specific antibodies.

Antibodies have been raised against N-cyclohexyl-N-(4-methylmorpholinium)ethyl carbodiimide (CMC) modified single-stranded DNA and characterized by competitive and non-competitive immunoassays to be highly specific for CMC base adduct in homopolymers poly(dG), poly(dT) and DNA. The antibodies recognize picogram concentrations of CMC treated DNA with no cross reactivity to at least 1000-fold excess of unmodified DNA or CMC treated poly(dA). The detection limit of antibodies at 1.4 fmol CMC adduct allows quantitation at a CMC/base ratio of 4.6.10(-7). Based upon single modified base-containing synthetic oligomers, a 7-fold higher binding preference is observed for CMC modified thymine than guanine bases. CMC binding to supercoiled DNA is found to depend upon reaction temperature and ionic strength. CMC-modified supercoiled SV40 and ColE1 DNA, exhibit specific antibody binding proportional to the DNA concentration and extent of CMC modification. However, antibody binding observed is independent of the conformation or strandedness of CMC-modified DNA. DNA extensively modified with CMC retains its inherent capacity to specifically and quantitatively hybridize with complementary DNA immobilized to membranes upon direct blotting or Southern transfers from gels. Hybridized CMC-DNA, through antibody binding, provides for the sensitive and non-isotopic detection of the target DNA sequences.

Expression of calf prochymosin in Saccharomyces cerevisiae.

A yeast strain which synthesizes activatable calf prochymosin (also known as prorennin) has been constructed by transformation with a vector carrying the methionyl-prochymosin coding sequence attached to efficient yeast transcriptional promoter and terminator sequences. Cloned preprochymosin cDNA was altered by restriction endonuclease cleavage and addition of a synthetic oligonucleotide to yield a DNA sequence encoding methionyl-prochymosin. This methionyl-prochymosin gene was ligated to a yeast chromosomal fragment containing the GAL1 promoter, and the construction was placed in an Escherichia coli-Saccharomyces cerevisiae shuttle vector with or without a transcriptional terminator DNA fragment from the yeast SUC2 gene. In yeast the two constructions result in equal amounts of prochymosin protein and mRNA. The prochymosin from yeast is activatable to chymosin by incubation at low pH and exhibits milk-clotting activity indistinguishable from calf chymosin.

Dendritic remodelling of retinal ganglion cells during development of the rat.

Investigation of the morphology of ganglion cells in the cat retina has shown that a remarkable reduction in the number of dendritic spines and branches occurs during development of the alpha and beta cell classes. To learn whether dendritic remodelling represents a generalized mechanism of mammalian retinal ganglion cell development, we have examined the morphology of ganglion cells in the retina of the developing rat. The present study has concentrated on type II cells, which retain a great number of dendritic spines and branches in the adult and comprise a large proportion of the population of rat retinal ganglion cells. To reveal fine dendritic and axonal processes, Lucifer yellow was injected intracellularly in living retinae maintained in vitro. Size and complexity of the dendritic trees were found to increase rapidly during an initial stage of development lasting from late fetal life until approximately postnatal day 12 (P12). Dendrites and axons of immature ganglion cells expressed several transient morphological features comprising an excessive number of dendritic branches and spine-like processes, and short, delicate axonal sidebranches. The following developmental stage was characterized by a remarkable decrease in the morphological complexity of retinal ganglion cells and a slowed growth of their dendritic fields. The number of dendritic branches and spines of types I and II retinal ganglion cells declined after P12 to reach a mature level by the end of the first postnatal month. Thus, even cells that retain a highly complex dendritic tree into the adult state undergo extensive remodelling. These results suggest that regressive modifications at the level of the dendritic field constitute a generalized mechanism of maturation in mammalian retinal ganglion cells.

Transient retinal ganglion cells in the developing rat are characterized by specific morphological properties.

To determine whether dendritic development of mammalian retinal ganglion cells (RGCs) is affected by axonal target specificity, the morphology of three populations of maturing RGCs was examined. These included RGCs that exhibited either a transient, topographically incorrect, projection to the caudal superior colliculus (SC), or a transient projection to the caudal inferior colliculus (IC), in addition to a control group that exhibited a topographically correct projection to the caudal SC. Projection populations were identified by retrograde transport of rhodamine labeled latex microspheres injected into target nuclei. Labeled RGCs were then injected in vitro with Lucifer yellow to reveal the details of their dendritic morphology. Retinal ganglion cells making target errors, most of which ultimately die, were found to undergo a remarkable degree of morphological differentiation and could be categorized according to the adult type I, II, or III criteria. However, the relative proportions of these cell types were different among RGCs making transient connections versus those whose projections were preserved. Approximately half of the RGCs making topographically incorrect projections to the SC belonged to type III, in contrast to 6% that made a topographically correct projection. In addition, the population of cells sending axons to caudal IC did not include type III RGCs, but consisted of small type II neurons. The development of the basic dendritic form of each RGC type was only modestly influenced by its projection pattern; dendritic trees of cells making transient projections were essentially normal with only a slight, but statistically significant, reduction in dimensions. Moreover, dendritic remodeling was evident during maturation of neurons making either transient or normal projections. Together, these findings indicate that target specificity plays a relatively minor role on dendritic development of retinal ganglion cells.

Developmental study of Müller cells in the rat retina using a new monoclonal antibody, RT10F7.

We produced the monoclonal antibody RT10F7, characterized its antigenic specificity and expression in the adult and developing retina, in cultured retinal cells and in other parts of the central nervous system. In metabolically-labelled retinal cultures RT10F7 immunoprecipitated a protein of approximately 36,000 mol. wt. In the adult, RT10F7 stained endfeet of Müller cells in the ganglion cell layer, four horizontal bands in the inner plexiform layer, and radial fibres in the outer plexiform layer which terminated at the outer limiting membrane. In the inner nuclear layer, most somata were underlined by Müller processes that wrapped around them, but some cell bodies were immunoreactive for RT10F7 in the cytoplasm. During development, postnatal day 21 was the first age at which the adult pattern of immunoreactivity was present, although a fourth band in the inner plexiform layer was less clear than for the adult. By 14 and eight days after birth, the pattern of RT10F7 immunoreactivity approximated that of the adult; however, only three bands and one band were present, respectively, in the inner plexiform layer. At earlier ages, postnatal days 4, 1 and embryonic ages 19 and 15, the monoclonal antibody stained Müller cell endfeet and radial fibres, from the inner plexiform layer through the neuroblastic layer to the outer limiting membrane. At these ages, the immunoreactivity was more prominent at the level of Müller cell endfeet. The monoclonal antibody stained glia in preparations of dissociated retinal cells maintained in culture but not astrocytes or oligodendrocytes from optic nerve cultures. In brain sections, tanycytes exhibited RT10F7 immunoreactivity. The monoclonal antibody RT10F7 recognized a specific cell type in the retina, the Müller cell. In the adult and developing retina, RT10F7 recognized an antigen that is present primarily in Müller cell processes. This feature allowed us to follow the maturation of the Müller cell and correlate it with developmental events in the retina. RT10F7 is a specific marker for Müller cells in vivo and in vitro and may be useful for studies of function of Müller cells after ablation or after injuries that are known to activate Müller cells.

Recognition of mitochondrial protein precursor lacking arginine at position -2 by mitochondrial processing peptidase: processing of bovine cytochrome P450(SCC) precursor.

Mitochondrial processing peptidase (MPP) specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Previous studies demonstrated that Arg at position -2 from the cleavage site, which is found among many precursors, plays a critical role in recognition by MPP. We analyzed the structural elements of bovine cytochrome P450 side-chain cleavage enzyme precursor [pre-P450(SCC)], which has Ala at position -2, for recognition by MPP. Replacement of Ala position -2 of pre-P450(SCC) with Arg resulted in an increase in the cleavage rate. Replacement with Gly caused a reduction in the cleavage rate and the appearance of an additional cleavage site downstream of the authentic site. A pre-P450(SCC) mutant with Met at position -2 retained cleavage efficiency equal to that of the wild type. These results indicate that -2 Ala of pre-P450(SCC) is recognized by MPP as a determinant for precise cleavage, and that the amino acid at -2 is required to have a straight methylene chain for interaction with the S(2) site. The preference for distal basic residues, a hydrophobic residue at +1, and hydroxyl residues at +2 and +3, was almost the same as those of the precursors with Arg at -2, indicating that the recognition mechanism of pre-P450(SCC) by MPP is essentially the same as that of the precursors with Arg at position -2.

Reduced nitric oxide production by L-arginine deficiency in lysinuric protein intolerance exacerbates intravascular coagulation.

Lysinuric protein intolerance (LPI) results in low serum L-arginine, hyperammonemia, mental retardation, thrombocytopenia, and an increased frequency of bowel movements. Our objective was to evaluate the effects of low serum L-arginine, the essential substrate for reactions catalyzed by nitric oxide synthetase (NOS), on the serum nitric oxide (NO) level and coagulation activity in a patient with LPI. A 37-year-old Japanese man who presented with abdominal pain and subnormal fasting levels of serum L-arginine and L-lysine was found to have LPI. The result of oral administration of diamino acids was an increased in urine and a decrease in serum, thus confirming the diagnosis. A decrease in the platelet count and an increase in the plasma levels of thrombin-antithrombin III complex (TAT) and fibrin degradation products (FDPs) indicated the presence of subclinical intravascular coagulation. Serum levels of NO derivatives and L-arginine were determined after intravenous administration of L-arginine. The effects of intravenous L-arginine or transdermal nitroglycerin on the plasma level of TAT were also investigated. Serum levels of NO derivatives were significantly reduced in the LPI patient versus the healthy control group (n = 5). Intravenous administration of L-arginine increased the serum level of NO derivatives and the platelet count and reduced plasma TAT and FDP levels. The plasma level of TAT was also reduced by transdermal nitroglycerin. A decrease in the serum level of L-arginine in patients with LPI appears to result in a decrease in NO production. The improvement in plasma TAT levels produced by administration of intravenous L-arginine or transdermal nitroglycerin suggests that intravascular coagulation is exacerbated by the decrease of NO production in patients with LPI.

Opposite roles of GABA and excitatory amino acids on the control of GAD expression in cultured retina cells.

The mechanism of control of GAD expression by GABA and excitatory amino acids (EAAs) was studied in chick and rat retina cultures using immunohistochemical and PAGE-immunoblot detection of the enzyme, as well as by measuring enzyme activity. Aggregate cultures were prepared with retina cells obtained from chick embryos at embryonic days 8-9 (E8-E9). Organotypical cultures were also prepared with retinas from E14 chick embryos, post-hatched chicken and P21 rats. GABA (1-20 mM) fully prevented GAD expression in aggregate and organotypical cultures from chick embryo retinas. A substantial, but not complete, reduction of GAD was also observed in organotypical cultures of post-hatched chicken and P21 rats, in which both forms of the enzyme (GAD65 and 67) were affected. The GABA effect was not mimicked by THIP (100 microM), baclofen (100 microM) or CACA (300 microM), agonists of GABAa, b and c receptors, respectively. NNC-711, a potent inhibitor of GABA transporters, reduced by 50% the inhibition of GAD activity promoted by GABA. Aggregates exposed to GABA and treated with glutamate (5 mM) or kainate (100 microM) displayed an intense GAD-like immunoreactivity in many cell bodies, but not in neurite regions. Immunoblot analysis revealed that the increase in GAD-like immunoreactivity by EAA corresponded to a 67-kDa protein. However, GAD activity was not detected. Treatment of aggregates or retina homogenates with SNAP, a NO producing agent (but not its oxidized form), reduced GAD activity by more than 60% indicating that the lack of enzyme activity in GAD-like immunoreactive cells, could be due to NO production by EAA stimulation.

GABAergic system in the developing mammalian retina: dual sources of GABA at early stages of postnatal development.

In the present work, we have characterized the maturation of the GABAergic system in mammalian retina. Immunoreactivity for GABA, GAD (glutamic acid decarboxylase, EC 4.1.1.15) -65 and -67 in the adult rat retina was localized in cells in the inner nuclear and ganglion cell layers. This pattern was established around postnatal day 8 and included transient GABA and GAD-67 expression in horizontal cells. GAD activity was very low at P1 and P4, increasing after P8, reaching maximal activity by P21 and decreasing to attain adult values by P30. GABA content was approximately constant from P1 to P13, increasing thereafter to reach adult levels. GAD protein content increased progressively with postnatal development and the two isoforms could be distinguished at P8. The disparity between retinal GABA content vs. presence and activity of the synthesizing enzyme, led us to investigate the alternative pathway for GABA synthesis that utilizes putrescine as a substrate. Highest levels of ornithine decarboxylase activity (the limiting step for putrescine synthesis) were found between P1 and P4, decreasing to very low levels after P13. The same pattern was observed for putrescine content in the retina. Highest amounts were found at P1, that decreased and remained constant after P13. Additionally, approximately 40% of tritiated putrescine incorporated by P1, P4 and adult retinas was converted into GABA. Our results suggest the existence of two different sources of GABA in mammalian retina, one that uses glutamate as a precursor and predominates in the mature nervous system and another that utilizes putrescine and is present transiently at early developmental stages.

Efficacy of combination therapy of interferon-alpha with ursodeoxycholic acid in chronic hepatitis C: a randomized controlled clinical trial.

The efficacy of interferon-alpha therapy in the treatment of chronic hepatitis C is still limited. A combination therapy of interferon-alpha with ursodeoxycholic acid (UDCA) was tested for its efficacy in the treatment of chronic hepatitis C by a randomized controlled study. Eighty consecutive Japanese patients with chronic hepatitis C were randomly divided into two groups: one group was treated with interferon-alpha (group A, n = 40) and the other with a combination of interferon-alpha and UDCA (group B, n = 40). In both groups, human interferon-alpha (6 million units per day) was intramuscularly injected daily for 2 weeks and then three times a week for 22 weeks: this 24-week period was followed by 24 weeks of observation. In group B, UDCA was also administered, daily at a dose of 600 mg orally, from the beginning of the interferon therapy and administration was continued for 48 weeks. The rates for ALT normalization and clearance of hepatitis C virus (HCV) viremia at the end of the 24-week interferon therapy were similar for groups A and B (58% vs 60% and 55% vs 48%, respectively). At the end of the 24-week follow-up, the sustained normalization rates for ALT levels for the two groups were not different (35% vs 43%), while the rate of clearance was higher in group B (40%) than in group A (23%), but the difference was not significant (P = 0.14). The sustained complete response, i.e., HCV RNA negativity at the end of the follow-up, as well as the maintenance of ALT normalization during the follow-up period, was more frequent in group B (38%) than in group A (18%) although the difference was not significant (P = 0.08). The rate of HCV reactivation after interferon was discontinued was significantly lower in group B (16%) than in group A (59%) (P < 0.01). Although this combination therapy did not lead to a sufficiently sustained complete response, it could serve as adjuvant antiviral therapy when a suitable dosage and administration period are determined.

A correction to the calculation of the Gibbs free energy of adsorption for biomolecules in ion-exchange systems.

We wish to propose a correction to the methodology introduced by Gerstner et al. [J.A. Gerstner, J.A. Bell, S.M. Cramer, Biophys. Chem. 52 (1994) 97-106] for the calculation of Gibbs free energies of adsorption of biomolecules to ion-exchange systems. Our approach is based on the requirement that the mobile phase and stationary phase concentrations be expressed in exactly the same units and the equilibrium constant be strictly dimensionless. The Gibbs free energies of ion-exchange calculated based on this correction appear to be more negative than those originally calculated by Gertner et al.

A new Salmonella tester strain, TA97, for the detection of frameshift mutagens. A run of cytosines as a mutational hot-spot.

We have developed a new Salmonella tester strain, TA97, for use in the Salmonella/microsome mutagenicity test. DNA sequencing has shown that this strain contains an added cytosine, resulting in a run of six cytosines at the mutated site in the histidine D gene. Its mutagenic specificity is similar to that of the frameshift mutagen tester strain, TA1537, which also contains an added cytosine in a run of cytosines and is currently among the five standard tester strains used for general mutagen screening. We assessed the mutagenic potency of 21 frameshift mutagens for TA1537 and TA97. TA97 was considerably more sensitive than TA1537 to reversion by these frameshift mutagens. In addition, one agent, PR toxin (from Penicillium roqueforti), which was not detected by any of the previously existing standard tester strains, did revert TA97; and two substituted aryl-alkyl triazenes, which had not been reported previously to be frameshift mutagens, were mutagenic in this new tester strain. We suggest that TA1537 be replaced by TA97 for general screening of mutagenicity.

Mutational activation of H-ras oncogene transformability by alkylnitrosourea-induced DNA damage.

To assess the role of DNA alkylation damage in oncogene activation, plasmid DNA containing H-ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with increasing concentrations of carcinogenic methylnitrosourea (MNU) and ethylnitrosourea (ENU). The modified plasmid DNA were analyzed by transfection-transformation of the NIH/3T3-recipient cells. Treatment with varying doses of MNU (0.1-5 mM) and ENU (1-15 mM) did not result in the inactivation of the plasmid containing target genes. A transformation efficiency of greater than 40% was observed upon treatment of H-ras oncogene with the highest doses of the alkylating agents. The morphologically transformed foci obtained with alkylated p220-EC ranged from 2.8 to 0.3/microgram MNU alkylated and 1.6 to 0.6/microgram ENU alkylated plasmid DNA. A significant proportion of the morphological transformants exhibited growth in soft agar. The HpaII/MspI restriction length polymorphism (RFLP) at codon 12 of H-ras exon-1 was detected with 4 independently isolated clones obtained from MNU-alkylated p220-EC transfections. Allele-specific in situ gel hybridization with a battery of codon 12 and codon 61 oligonucleotide probes confirmed these RFLPs to be due to sequence changes at codon 12. No clone with sequence changes in the H-ras codon 61 could be detected. The data indicate that a high degree of in vitro alkylation damage of the target gene is necessary to elicit mutational activation of H-ras in transfection-transformation assay. Low frequency notwithstanding, the data demonstrate that DNA alkylation damage at critical target sites can initiate neoplastic cellular transformation.

Continuous and discrete T-maze alternation in rats: effects of intertrial and interrun intervals.

1. Recent concepts concerning animal memory have emphasized the kind of information processed in memory. Reference memory provides information relevant over several trials, i.e., it codes expectancy-based information. Working memory provides information critical for only one trial, i.e., it codes data-based information. Some investigators consider that a continuous alternation task in a T-maze depends on the reference memory of a series of left-right responses, whereas a discrete alternation task is thought to depend on working memory. 2. In the present report, we tested rats in a continuous alternation task with different intertrial intervals (ITI's). Rats were first subjected to 10 or 12 sessions at each of the following ITI's: 0, 55, 100, 200 and 600 s, and then tested at varying ITI's within each session for 12 sessions in the following sequence: 0, 55, 100, 200, 600 and 0 s. Next, the same rats were trained to perform discrete alternation with ITI's and interrun intervals (IRI's) varying across sessions but only IRI's changing within sessions, i.e., IRI = 0 or 55 with ITI = 0, 55, 100, 200 or 600 s across sessions, and IRI = 0, 55, 100, 200 and 600 s with ITI = 0 within sessions. 3. Rats performed both alternation tasks at high levels when ITI's and IRI's changed across sessions. However, when intervals changed within sessions, rats showed a better performance in the continuous task at intervals of 55, 100 and 200 s compared to their performance at these same intervals in the discrete task. In addition, for the discrete task with IRI's changing within sessions, errors probably due to proactive interference occurred more frequently with progressively increasing IRI's. 4. The hypothesis that performance of continuous alternation depends on reference memory and performance of discrete alternation depends on working memory is supported by these data.

Chronic inflammatory demyelinating polyneuropathy accompanied by hepatocellular carcinoma.

We report a patient with chronic inflammatory demyelinating polyneuropathy (CIDP) accompanied by hepatocellular carcinoma (HCC). Due to the remarkable weakness in the lower limbs and loss of the position sense, he could not walk. On neurophysiological examination, he had impaired nerve conduction velocities. Biopsied nerve and muscle specimens demonstrated demyelination of nerve fibers and neurogenic degeneration of muscles. After steroid therapy he showed marked improvement in muscle strength and sensory function.

Granuloma leproide canino na região amazônica - relato de caso / Canine leproide granuloma in the amazon region - case report

Descreve-se o primeiro caso de granuloma leproide canino na região amazônica, Brasil, em um canino da raça Boxer, procedente do município de Castanhal, Pará, que apresentava lesões nodulares, alopécicas, firmes, ulceradas e não pruriginosas nas duas pinas. Os nódulos foram retirados cirurgicamente e enviados para análise histopatológica. O exame microscópico revelou marcada infiltração inflamatória constituída por macrófagos, plasmócitos, neutrófilos, linfócitos e células gigantes. A técnica de Ziehl-Neelsen evidenciou grande quantidade de bacilos álcool-ácido resistentes no interior de macrófagos e de células gigantes. Houve forte reatividade ao exame imuno-histoquímico para Mycobacterium spp.

This study describes the first case of canine leproid granuloma in the Amazon region, Brazil. A Boxer dog from the city of Castanhal, Pará presented nodular, alopecic, firm, ulcerated, non-pruritic lesions on both pinnae. The nodules were removed surgically and submitted to histopathological analysis. The microscopic exam revealed marked inflammatory infiltrate composed of macrophages, plasma cells, neutrophils, lymphocytes and giant cells. The Ziehl-Neelsen staining technique showed a large amount of alcohol-acid resistant bacilli inside macrophages and giant cells. The samples exhibited a strong immunohistochemical reaction to Mycobacterium spp.