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Bioactivation of quinones by DT-diaphorase, molecular, biochemical, and chemical studies.

Because of the elevated DT-diaphorase (DTD) activity in certain tumors such as human nonsmall cell lung cancer (NCSLC), DTD is a potential target on which to base the development of new antitumor compounds. Mitomycin C is the most effective single agent used for the therapy of NSCLC and is metabolized and bioactivated by DTD. Mitomycin C is a poor substrate for DTD, however, and its metabolism is pH-dependent. We have therefore focused on identifying more efficient substrates for DTD. We have developed a metabolic and cytotoxicity screen that identifies compounds which are efficiently bioactivated by DTD. This screen utilizes both aerobic and hypoxic conditions and cell lines with both elevated and deficient DTD activity as an index of selectivity. Using the screen described above, we have identified [3-hydroxy-5-aziridinyl-1-methyl-2-(1H-indole-4,7-indione)-prop-be ta-en- alpha-ol] (E09), 2,5-diaziridinyl-1,4-benzoquinone (MeDZQ), and streptonigrin as compounds that are most efficiently bioactivated by DTD and exert selective cytotoxicity. Although certain tumors such as NSCLC have elevated DTD activity, we have characterized a point mutation at position 609 in the DTD cDNA, which codes for a proline to serine change in the protein and leads to a loss of enzyme activity. We have characterized this mutation in both BE human colon carcinoma cells and H596 human NSCLC cells. This mutation and resulting lack of DTD activity complicates the use of agents designed to target DTD in tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Differences in haemagglutination activity and electrophoretic mobility of E protein between the parent SA14 and attenuated vaccine SA14-14-2 strains of Japanese encephalitis virus.

E protein characteristics of the attenuated Japanese encephalitis (JE) virus strain SA(A) derived from the live vaccine strain SA14-14-2 were compared with those of the virulent strain SA(V). SA(A) showed lower haemagglutination (HA) activity with broader optimum pH range of HA reaction than SA(V), and the E protein of SA(A) had slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than that of SA(V). These properties of SA(A) E protein appear to be caused by eight amino acid changes found in the E protein coding region of the SA(A) virus genome.

Amino Acids↗

Gene expression at graft-host interfaces of cortical bone allografts and autografts.

The healing processes of autogenous and frozen allogeneic cortical bone grafts were compared in rats. Two unicortical defects created in the proximal metaphysis of the tibia were autografted or allografted with blocks of cortical bone. The biomechanical properties of the graft-host junction and the time-related changes of the expression of genes coding for Type I, II, III, and X collagens and osteonectin were determined at one, two, four, and eight weeks. The strength of the allograft-host bone union was lower than that of autografts at the early stage of incorporation but the difference diminished by eight weeks. Northern analysis of graft mRNAs demonstrated a strong expression of Type I collagen and osteonectin genes at the beginning of autograft incorporation. The allografts showed a lower expression of the corresponding genes at one and two weeks, but the difference diminished thereafter. The gene coding for Type III collagen showed a temporary expression at two weeks both in allografts and autografts, probably because of the formation of inflammatory fibrovascular stroma provided by the host bone at the graft-host interface. No expression of cartilage-specific Type II and Type X collagens were observed, demonstrating that the healing in both grafts proceeded through primary bone healing. Compared with cortical-cortical junctions of autografts, the graft-host interfaces of allografts exhibited a reduced osteoinductive process and a slower increase of union strength.

Animals↗

Common variations in noncoding regions of the human natriuretic peptide receptor A gene have quantitative effects.

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.

Animals↗

Electronic and molecular structure of photoexcited [Ru(II)(bpy)3]2+ probed by picosecond X-ray absorption spectroscopy.

L(2,3) X-ray absorption spectra of aqueous [Ru(II)(bpy)3]2+ have been recorded in its ground and excited states, 50 ps after short pulse laser excitation. Significant changes in both the XANES (X-ray Near-Edge Absorption Structure) and the EXAFS (Extended X-ray Absorption Fine Structure) regions of the excited state complex are detected. The XANES line shapes have been quantitatively simulated using a crystal field multiplet code in trigonal symmetry. In addition, spectral changes in the EXAFS region of both ground and excited states are analyzed in order to extract structural parameters of their corresponding molecular structures. We obtain a Ru-N bond contraction by approximately 0.03 angstroms in the excited-state complex, as compared to the ground-state compound. This contraction results from electrostatic and polarization contributions, limited by steric constraints on the bpy ligands.

Journal Article↗

Neural control of motor output: can training change it?

Henneman's size principle of motor unit recruitment and rate coding reduces fatigue, minimizes error in transfer of information from the nervous system, and produces smooth force output. Plasticity present at various sites of the motor system may change endurance, force, speed, or precision with training, but not the recruitment order.

Exercise↗

Effects of horizontal cell network architecture on signal spread in the turtle outer retina. Experiments and simulations.

In the Pseudemys turtle retina five functionally distinct, electrically coupled networks of horizontal cells distribute signals in the outer plexiform layer. These networks differ significantly in their architecture, as determined by intracellular labeling with Neurobiotin after physiological recording and identification. The density of H1 horizontal cells is highest, ranging around 1800 cells/mm2 at approximately 2.3 mm eccentricity. H1 horizontal cell somata are connected via 6-10 thin, short dendrites. The H1 horizontal cell axon terminal network is composed of thick axon terminals, forming a three-dimensional, sheath-like structure. Networks of coupled H2 and H3 horizontal cells have cell densities of around 210 cells/mm2 and 350 cells/mm2, respectively, at the same eccentricity of 2.3 mm. Cell bodies are connected with 6-12 long, thin dendrites. Here we report for the first time H4 horizontal cell networks. Cell density is approximately 970 cells/mm2 at 2 mm eccentricity, and cell bodies are connected with 6-10 thin, short dendrites. General properties of passive voltage spread were compared for three of these horizontal cell networks using NeuronC. Realistic network architectures were obtained by digitizing the intracellularly labeled networks, respectively. One network obtained from coupled H1 horizontal cell bodies, one from coupled H1 horizontal cell axon terminals, and one from H2 horizontal cells were simulated. These three realistic networks were compared with an artificial, electrically coupled regular triangular network. Passive signal spread in these networks strongly depended on the exact network architecture using otherwise identical parameters. Changes in coupling strength affected signal spread in these networks differently. As in the experimental situation, changes in synaptic conductance influenced signal spread. Some principal effects of extensively coupled horizontal cells on photoreceptor signal processing were simulated with one type of photoreceptor connected by telodendria, synapsing onto an underlying triangular network and receiving feedback synapses. Under certain conditions, spatial information is coded in single photoreceptors. This was also the case in the experimental situation. In the simulation, spatial filter adjustment for optimal spatial coding in photoreceptors can be achieved by changing coupling strength in the horizontal cell network.

Animals↗

Genetic heterogeneity of the hepatitis C virus.

Hepatitis C virus (HCV) is an important etiological agent in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). The virus, identified only recently, contains a single-stranded RNA genome of positive polarity, is distantly related to pestiviruses and flaviviruses, and has been classified as the first member of a third genus within the family Flaviviridae. Extensive analysis of HCV genomic sequences demonstrated that this virus possesses significant genetic heterogeneity. Different regions of the viral genome demonstrate a varying degree of heterogeneity; the regions coding for the putative envelope proteins are the most variable sites between different isolates. Furthermore, HCV circulates as a quasispecies in the host. During the course of acute and chronic infection, the sequence composition of the HCV population in one patient has been found to change sequentially with an extremely high rate of nonconserved nucleotide changes in the hypervariable region I (HVR1) of HCV. Such sequence changes alter the antigenicity of the epitopes coded within HVR1 so that these are not always recognized by preexisting antibodies. It has been suggested that this could represent one mechanism by which HCV evades host immune surveillance and may account for the high rate of chronicity observed in such infections. Continuous viral replication may, in turn, lead to the development of chronic liver disease, including HCC, in infected individuals. To date, at least nine major genetic groups (genotypes 1-9) and more than 30 subgroups of HCV have been recognized based on genetic differences. A distinct difference has been observed in the genotype distribution in Africa compared with other continents. Recent data have suggested a difference in pathogenesis and in the outcome of interferon therapy in individuals infected with HCV of certain genotypes. For example, genotype 1b (II) seems to be associated with more severe liver disease, including HCC, and with a poorer response to interferon therapy. The extensive genetic heterogeneity of HCV may have serious implications for the diagnosis, treatment and prevention of hepatitis C as well as in understanding the biology of infection by this important human pathogen.

Genome, Viral↗

Molecular characterization of hprt mutants induced by low- and high-LET radiations in human cells.

Southern blotting techniques were employed to examine the spectrum of molecular alterations in DNA induced by internally emitting iodine isotopes and X-rays at and around the hprt locus in a human lymphoblastoid cell line. We analyzed 165 mutant clones using a cDNA probe for the human hprt locus, and 3 anonymous sequence probes for regions of the X chromosome which are linked to hprt. The results were compared with those for 35 spontaneously arising mutant clones. The majority of ionizing radiation-induced mutants showed changes in the normal restriction patterns at the hprt locus, whereas very few alterations were seen at linked markers along the X chromosome. Total hprt coding sequence deletions comprised 30-48% of the changes observed at this locus, while partial deletions and rearrangements comprised 14-54% of the observed changes. In the case of mutants induced by [125I]dUrd, a densely ionizing radiation, the spectrum of alterations was dose-dependent; at low doses it was not significantly different from that seen after sparsely ionizing X-ray exposure, whereas a higher proportion of gene deletions and rearrangements occurred after high doses of this incorporated isotope. Changes were rarely observed in the 3 linked markers examined. Overall, these results indicate that the distribution of mutational events at the hprt locus in irradiated human cells may not only be LET-dependent but dose-dependent, and that deletions involving large regions of the X chromosome surrounding the hprt locus are rare events.

Animals↗

Prevalences and correlates of ECG abnormalities in the adult Belgian population.

Data from 5,817 men and 5,215 women (age range, 25-74 years) who participated in the Belgian Interuniversity Research on Nutrition and Health were used to determine prevalences and associated variables of different abnormalities on the resting electrocardiogram. Electrocardiographic abnormalities were coded according to the Minnesota classification system. About one third of the subjects showed one or more abnormalities on their electrocardiogram. Major abnormalities occurred in 7.3% of the men and in 4.6% of the women. The prevalence of minor abnormalities was twice as high. Minnesota codes 4 (ST abnormalities) and 5 (T wave changes) were found to be the most prevalent in both sexes (9.6 and 10.5% respectively in women and 8.4 and 9.1% respectively in men), while code 2 (QRS axis deviation, 7.9%) and code 7 (ventricular conduction defect, 8.7%) were quite common in men. Most prevalences were rather log-linearly related with age, except codes 6 (atrioventricular conduction defect) and 9 (miscellaneous items). In the asymptomatic subjects, blood pressure showed the strongest positive correlation with minor ST-T abnormalities in both men and women independent of other factors. Other correlates identified by multivariate analyses were serum potassium, serum phosphor, and serum uric acid levels, as well as the intake of diuretics. However, any major, minor, and ST-T abnormalities were, according to sex, differently related to those correlates.

Adult↗

Sequence analysis of the EF-1 alpha gene family of Mucor racemosus.

Our previous studies have shown that Mucor racemosus possesses three genes (TEF-1, -2 and -3) for EF-1 alpha, and that all three genes are transcribed. However, the level of transcription varies markedly between the three genes, with TEF-1 mRNA levels being approximately two fold higher than TEF-3 and 6 fold higher than TEF-2. We have now completed the DNA sequence of both strands of all three genes and have found that these genes are highly homologous. TEF-2 and TEF-3 are more similar to each other than they are to TEF-1. The TEF-2 and the TEF-3 coding regions differ from TEF-1 at 30 and 37 positions respectively out of 1374 nucleotides. Twenty-six of these nucleotide substitutions were common to both TEF-2 and TEF-3, and the majority of the substitutions were clustered in the 5' region of the coding sequences. While the majority of these changes were silent, TEF-2 and TEF-3 differed from TEF-1 by having a lysine instead of a glutamate at amino acid position 41. In addition, TEF-2 and -3, but not TEF-1, each have an intron located near the 5' end of the coding region, although its size and sequence is not conserved between the two genes. All three genes have a conserved intron near the 3' end of the coding region. The sequence data have been analyzed with respect to the structure and function of EF-1 alpha in protein biosynthesis.

Amino Acid Sequence↗

Asthma deaths in the United Kingdom.

Mortality due to asthma was recognised by Thomas Willis in 1671 though it was not until the early 1960s that an epidemic of death raised major concern in the United Kingdom. Initially over-treatment with beta agonist aerosols was blamed as the cause both here and in Australia and New Zealand. Further studies in London, Cardiff and Birmingham suggested this was not the case, rather than under diagnosis, under appreciation and under treatment were a more common cause of asthma mortality. The British Thoracic Society's confidential survey in 1979 confirmed these findings and further suggested that wrong certification was a potential source of inaccuracy in older age groups. More recently the New Zealand Asthma Task Force have reported the results of a two year national survey of deaths, revealing similar results to the UK studies, also finding little evidence of beta agonist over usage, little effect of nebulized drug administration and no significant theophylline drug over usage. An examination of trends in Asthma Mortality in the United Kingdom by Stewart and Nunn shows that the changes in the World Health Organisation I.C.D. coding of 1967 and 1978 had little overall effect on asthma death certification in the 15-44 year old age group. When this is taken into account they found that asthma mortality has declined from 1958 to 1982 in females but that there has been no change in males despite therapeutic advances over the period. Asthma mortality date derived since; however, does suggest a small but significant annual incremental rise in the 15 to 44 year age group.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression.

Little is known about the role of genetic and epigenetic control in the spatial and temporal regulation of plant development. Overexpressing antisense Arabidopsis thaliana HD1 (AtHD1) encoding a putative major histone deacetylase induces pleiotropic effects on plant growth and development. It is unclear whether the developmental abnormalities are caused by a defective AtHD1 or related homologs and are heritable in selfing progeny. We isolated a stable antisense AtHD1 (CASH) transgenic line and a T-DNA insertion line in exon 2 of AtHD1, resulting in a null allele (athd1-t1). Both athd1-t1 and CASH lines display increased levels of histone acetylation and similar developmental abnormalities, which are heritable in the presence of antisense AtHD1 or in the progeny of homozygous (athd1-t1/athd1-t1) plants. Furthermore, when the athd1-t1/athd1-t1 plants are crossed to wild-type plants, the pleiotropic developmental abnormalities are immediately restored in the F(1) hybrids, which correlates with AtHD1 expression and reduction of histone H4 Lys12 acetylation. Unlike the situation with the stable code of DNA and histone methylation, developmental changes induced by histone deacetylase defects are immediately reversible, probably through the restoration of a reversible histone acetylation code needed for the normal control of gene regulation and development.

Arabidopsis↗

Proper coding and billing for office services.

The information revolution has effected tumultuous change in the process by which physicians receive compensation for their services. In an effort to predict and control costs, a labyrinthine coding system, together with multilayered rules and regulations, has been developed to pinpoint medical function. Computerization of the insurance industry allows vigilant scrutiny of the physicians claims. Since noncompliance with payment rules now carries legal or monetary consequences, physicians need to pay careful attention to payer methodology, not only to fulfill the requirements of the law but also to receive accurate and optimal reimbursement. This article is designed to help practitioners unscramble these exacting mandates and stay in alignment.

Fees and Charges↗

Leishmania braziliensis, molecular characterization of an elongation factor 1alpha gene.

The elongation factor EF-1alpha is one of the most studied components of the translation machinery owing to its abundance and possible role in other cellular functions. EF-1alpha mediates the correct coupling of the aminoacyl-tRNA on the A site of the ribosome in a GTP-dependent reaction. We have previously described an EF-1alpha DNA sequence in Leishmania amazonensis, pLEF11 (accession No. M92653), using PCR. In this paper we describe the DNA sequence and genomic organization of L. braziliensis EF-1alpha gene. Southern blot analysis revealed that EF-1alpha is organized as a 2 kb tandem repeat. The pLEF11 probe recognized a 1.8 kb mRNA from promastigotes in Northern blots. A clone containing the first copy and a half of the EF-1alpha tandem repeat was isolated by screening a L. braziliensis genomic library. Southern blot analysis showed that the isolated clone (lambda2.2) presented the same hybridization profile as that of a genomic blot. The partial sequencing of clone lambda2.2 spans 2959 nucleotides in length, which has two open reading frames separated by a putative non-coding region. The nucleotide and the predicted peptide sequence of the first coding region presented approximately 80% identity with other eukaryotic EF-1alpha genes. The sequence also displayed the four consensus motifs corresponding to the GTP-binding site (G1, G2, G3 and G4). Computer analysis of the sequence of both coding regions revealed three divergent nucleotides, which generated two changes at the amino acid level. One was found to be located in the G2 domain. The non-coding region of the EF-1alpha gene sequence showed potential regulatory elements such as polypyrimidine tracks, chi-homologous sequences and stem-loop forming sequences.

Amino Acid Sequence↗

Postoperative elevation of creatine kinase (CK-MB): does it contribute to diagnosis of myocardial infarction?

PURPOSE: This retrospective study sought to determine the benefit of measurement of changes in plasma creatine kinase-myocardial band (CK-MB) levels in elective postoperative high risk surgical patients beyond that obtained from the surface 12 lead ECG. METHODS: The charts of 111 patients admitted to the surgical intensive care unit (SICU) of a tertiary level university teaching hospital were reviewed. They were screened using predetermined definitions of myocardial infarction (MI) (as reflected by changes in the 12 lead surface ECG (Minnesota code) or elevations in CK-MB) for complications such as pulmonary oedema, congestive heart failure, arrhythmias, or cardiogenic shock. Four groups were identified based on changes in the ECG indicative of MI (Present-ECG+ or Absent-ECG-) and elevations of CK-MB (Present-CKMB+ or Absent-CKMB-) and compared for the incidence of complications. RESULTS: No patient with ECG- findings had a complication. Fifteen patients with ECG+ findings were identified and all had complications. Fourteen of these patients had CKMB+ results. In contrast, 29 patients with CKMB+ results alone (i.e., ECG-) had no complications. CONCLUSION: Clinically important (i.e., requiring therapeutic intervention) postoperative myocardial infarction was detected by ECG changes. The benefit of determining changes in CK-MB was minimal from a therapeutic perspective.

Adult↗

Comparative transcriptomics of Venus flytrap (Dionaea muscipula) across stages of prey capture and digestion.

The Venus flytrap, Dionaea muscipula, is perhaps the world's best-known botanical carnivore. The act of prey capture and digestion along with its rapidly closing, charismatic traps make this species a compelling model for studying the evolution and fundamental biology of carnivorous plants. There is a growing body of research on the genome, transcriptome, and digestome of Dionaea muscipula, but surprisingly limited information on changes in trap transcript abundance over time since feeding. Here we present the results of a comparative transcriptomics project exploring the transcriptomic changes across seven timepoints in a 72-hour time series of prey digestion and three timepoints directly comparing triggered traps with and without prey items. We document a dynamic response to prey capture including changes in abundance of transcripts with Gene Ontology (GO) annotations related to digestion and nutrient uptake. Comparisons of traps with and without prey documented 174 significantly differentially expressed genes at 1 hour after triggering and 151 genes with significantly different abundances at 24 hours. Approximately 50% of annotated protein-coding genes in Venus flytrap genome exhibit change (10041 of 21135) in transcript abundance following prey capture. Whereas peak abundance for most of these genes was observed within 3 hours, an expression cluster of 3009 genes exhibited continuously increasing abundance over the 72-hour sampling period, and transcript for these genes with GO annotation terms including both catabolism and nutrient transport may continue to accumulate beyond 72 hours.

Droseraceae↗

A molecular biologic study of extracellular matrix components during the development of glomerulosclerosis in murine chronic graft-versus-host disease.

BACKGROUND: We studied the development of glomerulosclerosis in murine chronic graft-versus-host disease, a model for human systemic lupus erythematosus. EXPERIMENTAL DESIGN: The disease was induced in (C57BL10 x DBA/2)F1 hybrids by injection of DBA/2 lymphocytes leading to deposition of auto-antibodies in the glomeruli, and a lupus type of nephritis morphologically. We have determined the levels of mRNA coding for laminin (B1 and B2), a 67 kilodalton laminin binding protein, and types I and IV collagen, in control and graft-versus host disease mice at various times after disease induction. RESULTS: Laminin and collagen mRNAs were increased in whole kidneys 4 weeks after induction of the disease. At week 10, all animals displayed dramatic stimulation of alpha 1(I), alpha 1(IV), laminin B1, and B2 mRNAs. The 67 kilodalton laminin binding protein mRNA was also doubled from week 4 to 16. In isolated glomeruli, the mRNA level coding for laminin B2 was already significantly increased from week 8. This enhancement of laminin synthesis corresponds to the mesangial expansion and to the development of laminin-containing spike formations of the glomerular basement membrane at week 8. CONCLUSIONS: The expansion of the mesangial matrix in murine chronic graft-versus-host disease is caused at least in part, by an increased production of extracellular matrix components by glomerular cells. These results demonstrate that the increase of specific extracellular matrix components mRNAs precedes light microscopic changes. Quantitative evaluation of the mRNA levels coding for extracellular matrix proteins may reveal a useful method for the early detection of the development of glomerular sclerosis at the stage preceding the onset of anatomo-clinical changes.

Animals↗