Cyanide-induced transition from endogenous carbohydrate to lipid oxidation as indicated by the carbon-13 content of respiratory CO2

The respiration of thin aerated discs of potato tuber tissue rises sigmoidally through 24 h. Aged disc respiration is ostensibly resistant to concentrations of cyanide which inhibit the respiration of fresh discs. It has been shown that cyanide-resistant respiration does not represent indifference to the inhibitor, but is rather due to the suppression of one respiratory carbon path and the evocation of another. The predominant respiratory carbon path of aged discs in the absence of cyanide comprises glycolysis linked to the tricarboxylic acid cycle. The carbon path mediating the cyanide-induced respiration reflects tricarboxylic acid cycle-independent lipid degradation.

The respiratory substrate at any time was deduced by comparing the ¹³C/¹²C ratio of respired CO₂, collected from discs in the presence or absence of cyanide, with the ¹³C/¹²C ratios characterizing endogenous potential metabolites. The determination of the predominant respiratory substrate in potato discs, which have an endogenous substrate reserve, proved possible because the relative concentrations of the stable carbon isotopes in endogenous compounds such as lipid and starch are widely different.     

Delta opioid binding selectivity of 3-ether analogs of naltrindole

Masking of the 3-phenol of naltrindole as a range of ethers caused a decrease in binding affinity at all three opiate receptors (μ, κ, δ), however for the methyl ether, the reduction in affinity at both μ and κ was greater than at δ, thereby increasing δ binding selectivity.     

Evaluation of an in vitro coculture model for the blood-brain barrier: Comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources

Summary Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.     

Molecular Evolution of the Human Enteroviruses: Correlation of Serotype with VP1 Sequence and Application to Picornavirus Classification

Sixty-six human enterovirus serotypes have been identified by serum neutralization, but the molecular determinants of the serotypes are unknown. Since the picornavirus VP1 protein contains a number of neutralization domains, we hypothesized that the VP1 sequence should correspond with neutralization (serotype) and, hence, with phylogenetic lineage. To test this hypothesis and to analyze the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 sequences of the prototype strains of 47 human enterovirus serotypes and 10 antigenic variants. Our sequences, together with those available from GenBank, comprise a database of complete VP1 sequences for all 66 human enterovirus serotypes plus additional strains of seven serotypes. Phylogenetic trees constructed from complete VP1 sequences produced the same four major clusters as published trees based on partial VP2 sequences; in contrast to the VP2 trees, however, in the VP1 trees strains of the same serotype were always monophyletic. In pairwise comparisons of complete VP1 sequences, enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15 viruses should be classified as strains of the same serotype, as should coxsackie A13 and A18 viruses. Pairwise identity scores also distinguished between enteroviruses of different clusters and enteroviruses from picornaviruses of different genera. The data suggest that VP1 sequence comparisons may be valuable in enterovirus typing and in picornavirus taxonomy by assisting in the genus assignment of unclassified picornaviruses.Human enteroviruses (family Picornaviridae) infect millions of people worldwide each year, resulting in a wide range of clinical outcomes ranging from inapparent infection to mild respiratory illness (common cold), hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis (reviewed in references 43 and 45). In the United States, enteroviruses are responsible for 30,000 to 50,000 meningitis hospitalizations per year as a result of 30 million to 50 million infections. Serologic studies have distinguished 66 human enterovirus serotypes on the basis of an antibody neutralization test (43), and additional antigenic variants have been defined within several of the serotypes on the basis of reduced or nonreciprocal cross-neutralization between prototype and variant strains (6, 8, 68, 71, 72). On the basis of their pathogenesis in humans and experimental animals, the enteroviruses were originally classified into four groups, polioviruses, coxsackie A viruses (CA), coxsackie B viruses (CB), and echoviruses, but it was quickly realized that there were significant overlaps in the biological properties of viruses in the different groups (8). The more recently isolated enteroviruses have been named with a system of consecutive numbers: EV68, EV69, EV70, and EV71 (42).A comparison of nucleotide and deduced amino acid sequences at the 5′ end of VP2 has identified four major phylogenetic groups within the Enterovirus genus: CA16-like viruses (cluster A), a CB-like group containing all CB and echoviruses as well as CA9 and EV69 (cluster B), poliovirus-like viruses (cluster C), and EV68 and EV70 (cluster D) (23, 24, 49, 53, 54, 73). However, pairwise alignments and phylogenetic analyses within these groups demonstrated that the VP2 sequence does not fully correlate with serotype, as viruses known to belong to the same serotype often failed to cluster together (2, 49). (E22 and E23 are genetically distinct from enteroviruses [24], and their reclassification into a separate genus has been proposed [45]).VP1 is the most external and immunodominant of the picornavirus capsid proteins (58). A number of major neutralization sites reside in the VP1 proteins of many picornaviruses (reviewed in references 40 and 44), but the specific epitopes responsible for serotype specificity and intratypic variation have not been identified. Similarly, the genetic correlates of serotype identity remain unknown. If the important serotype-specific neutralization sites reside in VP1, then the VP1 sequence or some portion thereof would be predicted to correlate with serotype. Studies on the three serotypes of poliovirus have shown that a partial VP1 sequence correlates well with serotype (32). In addition, genetic lineages based on the VP1 sequence can be used to define poliovirus reservoirs and chains of transmission (reviewed in reference 30). To test whether the VP1 sequence might be applied to the classification of nonpolio enteroviruses and to the analysis of the phylogenetic relationships among the human enteroviruses, we determined the complete VP1 nucleotide sequences for 47 human enterovirus prototypes and 10 well-characterized antigenic variants. These data, together with previously available sequences, comprise a database of complete VP1 sequences for all known human enterovirus serotypes and 12 natural antigenic variants. This database will be useful for molecular epidemiologic studies of enteroviral disease outbreaks, to obtain a better understanding of the genetic correlates of serotype, and for the development of enteroviral molecular diagnostic reagents.     

Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated “core” sequences (GACA, GATA, and GCAC). The principal objectives of this investigation were to determine whether: (1) the previously reported almost complete lack of polymorphism at allozyme loci in this species was indicative of a reduced amount of genetic diversity at VNTR loci as well; (2) VNTR markers were informative about possible clonal propagation; and (3) significant differences in genetic structure of sampling sites were associated with differences in environmental levels of pollutants at those sites. Previously, widespread sampling across the eastern United States, surveying across ten allozyme loci, has detected only two genotypes, involving a difference at a single locus, among 104 populations. In this study, the amount of genetic diversity detected at VNTR loci: (1) among ramets (N = 40; 40 genotypes detected) collected at ∼8-km intervals along a 320-km transect; (2) among ramets (N = 220; 117 genotypes detected) from five study sites separated by 50–3000 m; and (3) even among ramets within each study site [N = 44 per site; from 13 to 34 genotypes detected per site (270 m²)] exceeds that previously found in those more geographically widespread allozyme surveys. Among the 260 ramets analyzed here, the mean number of bands scored per individual was 48.61 (SD = 2.80). Mean genetic similarity among ramets collected along the 320-km transect was 0.91, which was within the range of mean genetic similarity within the five study sites (range: 0.89–0.95). Among the five study sites, 61% of the samples analyzed appeared to be clonal ramets, with up to 12 clones detected for 44 ramets sampled within a site. Clones grew intermingled and ranged up to 39 m in extent. Permutation tests of genetic similarity revealed significant genetic differentiation between each of the five study sites. Consistent with the previous allozyme studies, T. latifolia was characterized by extremely low genetic variation relative to levels of polymorphism detected at VNTR loci in other plant species. Estimated heterozygosity among ramets along the 320-km transect ranged from 0.11 to 0.13, while that within the five study sites ranged from 0.05 to 0.12. Estimates of F_st (0.32–0.41) also indicated considerable genetic subdivision among these stands. Significantly higher genetic diversity was detected at the two study sites that chemistry and toxicity data indicate to be the most severely impacted by pollutants. Although this correlation does not establish cause and effect, the results of this study indicate that the analysis of genetic diversity at VNTR loci may be a useful tool for monitoring anthropogenic-induced changes in the genetic structure of natural populations of plants.     

Sugar meals and longevity of the sandfly Phlebotomus papatasi in an arid focus of Leishmania major in the Jordan Valley

The sugar diet and life-span of Phlebotomus papatasi were studied in a typical zoonotic focus of Leishmania major in an arid area of the Jordan Valley during 1996-1997. Plant-tissue residues (cellulose particles) were identified in the stained guts of 23% of P. papatasi and significant amounts of sugar were found in the gut of 16%. Feeding on different plants was demonstrated by using their branches, suffused with cellulose stain, as baits in the field. Ingested, stained cellulose was detected in 10% of the sandflies (6% of males, 12.5% of females) caught near bait-branches of common local plants, mostly Chenopodiaceae. The similar rates of plant and sugar feeding, with the observed absence of aphids (ruling out the availability of honeydew), implied that the sugar meals of sandflies were obtained directly from plants. The relative paucity of sugar meals in P. papatasi coincided with a short life-span, evaluated by daily growth lines in the cuticle. The age of the oldest females was estimated to be 8 days, and 6 days for males. Under local conditions, the first gonotrophic cycle can be completed in 6 days and the usual transmission of L. major is apparently afterwards, when females ingest blood to initiate another cycle. Only about 9% of P. papatasi females survived > 6 days.     

Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg²⁺) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na⁺ pump andCl channels. In this study,we determined whether theNa⁺-K⁺-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg²⁺ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg²⁺inhibitsNa⁺-K⁺-Clcotransport, with inhibitor constant(K_i) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg²⁺ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg²⁺binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

     

Association of immune parameters with clinical outcome in stage III colon cancer: results of Southwest Oncology Group Protocol 9009

Levamisole (LMS), utilized in the adjuvant treatment of patients with stage III colon cancer, is immunomodulatory. To determine whether alterations in immune parameters before, during and after 12 months of 5FU/LMS therapy correlate with disease-free survival, 38 patients enrolled on Southwest Oncology Group (SWOG) protocol 8899 received extensive lymphocyte phenotypic analysis prior to therapy and 3, 6, 12 and 15 months after treatment initiation. The median follow-up of patients is 41 months. Significant increases in the proportion and total number of CD56⁺ natural killer cells were seen, starting at 3 months and continuing until 15 months (P < 0.001). Increases in the total numbers of cells expressing CD25 (interleukin-2 receptor), VLA4 and the combinations of CD4: CD45RA and CD4:CDw29 were not evident during therapy but were seen at 15 months (P < 0.05: CD25, CD4:CDw29, CD4:CD45RA; P < 0.001: VLA4). Low levels of CD8⁺ cells prior to treatment initiation and after 3 months of therapy correlated with early relapse within the first year of 5FU/LMS treatment. Patients who have remained disease-free (n = 22, median follow-up 45 months) demonstrated increases in the total numbers of CD8⁺, CD25⁺, CD56⁺, VLA4⁺, CD4: CDw29 and CD4:CD45RA cells, primarily at 15 months. In contrast, patients who relapsed had decreased numbers of CD8⁺, CD4:CDw29, CD4: CD45RA and VLA4⁺ cells and minimal increases in CD56⁺ and CD25⁺ cells. Statistically significant differences between the late-relapse group and the group remaining disease-free were seen for CD25⁺, CD4: CD45RA and CD4:CDw29 cells at the 15-month assay time (P = 0.0276, P = 0.0349, P = 0.0178 respectively). In conclusion, multiple alterations in lymphocyte phenotype, with increases in the proportion and total number of cells involved in cell-mediated immune responses, were seen during and especially following completion of therapy with 5FU/LMS. Many of these changes are significantly associated with clinical outcome and may be useful for risk stratification of stage III colon cancer patients following completion of adjuvant therapy. Received: 9 July 1999 / Accepted: 11 August 1999     

Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells

The objective ofthis study was to assess the effects of two structurally distinct yetselective proteasome inhibitors (PS-341 and lactacystin) on leukocyteadhesion, endothelial cell adhesion molecule (ECAM) expression, andnuclear factor-B (NF-B) activation in tumor necrosisfactor (TNF)--stimulated human umbilical vein endothelial cells(HUVEC) and the transformed, HUVEC-derived, ECV cell line. We foundthat TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclearneutrophil (PMN) adhesion to HUVEC but not to ECV; TNF alsosignificantly enhanced surface expression of vascular cell adhesionmolecule 1 and E-selectin (in HUVEC only), as well as intercellularadhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVECwith lactacystin completely blocked TNF-stimulated PMN adhesion,partially blocked U-937 adhesion, and completely blocked TNF-stimulatedECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1expression in ECV. Pretreatment of HUVEC with PS-341 partially blockedTNF-stimulated leukocyte adhesion and ECAM expression. These effects oflactacystin and PS-341 were associated with inhibitory effects onTNF-stimulated NF-B activation in both HUVEC and ECV. Our resultsdemonstrate the importance of the 26S proteasome in TNF-inducedactivation of NF-B, ECAM expression, and leukocyte-endothelialadhesive interactions in vitro.