Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides

N(1)-Phenyl-3,5-dinitro-N(4),N(4)-di-n-propylsulfanilamide (1) and N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (2) show potent in vitro antimitotic activity against kinetoplastid parasites but display poor in vivo activity. Seventeen new dinitroaniline sulfonamide and eleven new benzamide analogs of these leads are reported here. Nine of the sulfonamides display in vitro IC(50) values under 500 nM against African trypanosomes, and the most active antikinetoplastid compounds also inhibit the in vitro assembly of purified leishmanial tubulin with potencies similar to that of 2. While several of the potent compounds are rapidly degraded by rat liver S9 fractions in vitro, N(1)-(3-hydroxy)phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (21) displays an IC(50) value of 260 nM against African trypanosomes in vitro and is more stable than 2 in the in vitro metabolism assay.     

Correlation of genomic and physiological traits of thermoanaerobacter species with biofuel yields

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B₁₂ biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.     

Contrasting population characteristics of yellow bass (Morone mississippiensis) in two southern Illinois reservoirs

Two southern Illinois reservoirs were investigated with contrasting size structures of yellow bass (Morone mississippiensis) to compare growth, mortality and recruitment patterns. Yellow bass were collected from Crab Orchard and Little Grassy lakes during April–May 2009 using AC electrofishing. Total length and weight were recorded and sagittal otoliths sectioned and aged by two readers. Increments between otolith annuli were measured and the Weisberg linear growth model was used to assess age and environmental (growth year) effects on individual growth for fish from the two lakes. Von Bertalanffy growth models indicated faster growth and a greater maximum total length for yellow bass in Little Grassy Lake. However, growth of fish in Little Grassy Lake nearly ceased after age 4. The Weisberg model indicated differences in individual growth rate between the two lakes that were consistent across years (age effects were significant, but growth year effects and the age‐growth year interaction were not). Inter‐lake differences in fish growth were present up to age 3. Recruitment was relatively stable in Crab Orchard, with year classes up to age 7 observed. Recruitment was more erratic in Little Grassy, with age 5 being the dominant year class and fish up to age 11 present. Differences in growth and recruitment patterns for yellow bass in the two lakes may be attributed to substantial inter‐lake differences in turbidity, morphoedaphic index, or yellow bass density. Maximum age of yellow bass (age 11) was higher than previously reported for this species, likely due to the use of otoliths to age fish rather than scales. This study provides baseline information on age and growth, mortality, recruitment, and size structure of yellow bass that can be compared to data in future studies to elucidate factors influencing population dynamics of this species.     

A novel method for measuring medial compartment pressures within the knee joint in-vivo

A novel method for the measurement of knee joint forces in-vivo is described. A thin (0.2mm) flexible electronic pressure sensor was inserted through a narrow arthroscopic portal into the osteoarthritic medial compartment of the knee joint. The sensor partially covered the load bearing area. The surgery was performed under local anaesthetic during normal arthroscopic examination following patient consent. Results are presented for 11 patients. The method was used in a pilot study to assess the effects of four valgus knee braces on medial compartment forces. An analysis of variance could not detect un-loading by any brace although there were large variations in force output. These variations may be attributable to shifts in the sensor position. In-vivo measurement of joint force is technically feasible.     

N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain.

N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with /=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.     

Expression and characterization of a chimeric bifunctional antibody with therapeutic applications

A simple method is described for the generation of a biologically produced mouse/human chimeric hetero-bifunctional antibody that has dual specificity for human carcinoembryonic Ag and metal chelate haptens. Two large compound chimeric vectors each containing the genetic information to produce a single antibody specificity were sequentially electroporated into the murine nonsecreting hybridoma SP2/0. This led to the isolation of a clone expressing high levels of total IgG (up to 25 micrograms/ml/10(6) cells), 10 to 20% of which showed simultaneous reactivity with both Ag. Binding studies showed that the immunoreactivities and affinity constants for the individual arms of the bifunctional antibody were equivalent to those seen with the parental antibodies.     

Fine needle aspiration cytology of pilomatrixoma (calcifying epithelioma of Malherbe). Report of a case.

A case of a facial pilomatrixoma (calcifying epithelioma of Malherbe) in a child initially diagnosed following fine needle aspiration is presented. On cytologic examination, basaloid cells, multinucleated giant cells and deposits of calcium were identified. Adjacent to the basaloid cells were acellular masses suggestive of, but not diagnostic of, ghost cells. The diagnosis was confirmed by histology.     

Genetic variation in the developmental regulation of cortical avpr1a among prairie voles

Early experiences can have enduring impacts on brain and behavior, but the strength of these effects can be influenced by genetic variation. In principle, polymorphic CpGs (polyCpGs) may contribute to gene‐by‐environment interactions (G × E) by altering DNA methylation. In this study, we investigate the influence of polyCpGs on the development of vasopressin receptor 1a abundance in the retrosplenial cortex (RSC‐V1aR) of prairie voles (Microtus ochrogaster). Two alternative alleles (‘HI’/‘LO’) predict RSC avpr1a expression, V1aR abundance and sexual fidelity in adulthood; these alleles differ in the frequency of CpG sites and in methylation at a putative intron enhancer. We hypothesized that the elevated CpG abundance in the LO allele would make homozygous LO/LO voles more sensitive to developmental perturbations. We found that genotype differences in RSC‐V1aR abundance emerged early in ontogeny and were accompanied by differences in methylation of the putative enhancer. As predicted, postnatal treatment with an oxytocin receptor antagonist (OTA) reduced RSC‐V1aR abundance in LO/LO adults but not their HI/HI siblings. Similarly, methylation inhibition by zebularine increased RSC‐V1aR in LO/LO adults, but not in HI/HI siblings. These data show a gene‐by‐environment interaction in RSC‐V1aR. Surprisingly, however, neither OTA nor zebularine altered adult methylation of the intronic enhancer, suggesting that differences in sensitivity could not be explained by CpG density at the enhancer alone. Methylated DNA immunoprecipiation‐sequencing showed additional differentially methylated regions between HI/HI and LO/LO voles. Future research should examine the role of these regions and other regulatory elements in the ontogeny of RSC‐V1aR and its developmentally induced changes.