A phosphorylated prodrug for the inhibition of Pin1

Fmoc-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole 1, showed moderate inhibition towards the mitotic regulator, Pin1 (IC(50)=28.3microM). To improve the cell permeability, the charged phosphate was masked as the bis-pivaloyloxymethyl (POM) phosphate in Fmoc-(bisPOM)-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole 2. Antiproliferative activity towards A2780 ovarian cancer cells of 1 (IC(50)=46.2microM) was improved significantly in 2 (IC(50)=26.9microM), comparable to the IC(50) of 1 towards Pin1 enzymatic activity.     

Seasonal changes in fish catch and environmental variables in a large Tropical Lake,Volta, Ghana

Fish species assemblage and selected environmental variables were monitored in Lake Volta from September 2014 to August 2016 to determine seasonal variability in species composition, catch and environmental variables that determine the structure of the fish community. A total of 1,557 individual fish belonging to 41 species, 25 genera and 13 families were recorded. The important fish species with respect to frequency of occurrence, abundance and weight, respectively, were as follows: Chrysichthys (100%; 43.03%; 17.93%), tilapias (100%; 28.97%; 17.86%), Alestes (100%; 14.13%; 32.10%) and Bagrus (91.67%; 5.65%; 12.80%) in that order. The composition of fish species and their temporal variation in experimental catches were similar to that of commercial catches. There were no significant differences (p > 0.05) in species abundance, weight and diversity indices between the dry and wet seasons. The modal class of length frequency distribution of the dominant species in the catch, Chrysichthys, reduced from 125 mm in 2006 to 95 mm in the current study indicating overfishing. Environmental variables considered showed little variation and within optimal ranges for fish survival and did not differ significantly between seasons. Canonical Correspondence Analysis showed that environmental variables explained 43.30% of the variation in species abundance with Lake water level, nitrite‐nitrogen and total dissolved solids being the main environmental factors influencing the structure of the fish community.     

An Experimental Test of Competition among Mice,Chipmunks, and Squirrels in Deciduous Forest Fragments

Mixed hardwood forests of the northeast United States support a guild of granivorous/omnivorous rodents including gray squirrels (Sciurus carolinensis), eastern chipmunks (Tamias striatus), and white-footed mice (Peromyscus leucopus). These species coincide geographically, co-occur locally, and consume similar food resources. Despite their idiosyncratic responses to landscape and patch variables, patch occupancy models suggest that competition may influence their respective distributions and abundances, and accordingly their influence on the rest of the forest community. Experimental studies, however, are wanting. We present the result of a large-scale experiment in which we removed white-footed mice or gray squirrels from small, isolated forest fragments in Dutchess County, New York, and added these mammals to other fragments in order to alter the abundance of these two species. We then used mark–recapture analyses to quantify the population-level and individual-level effects on resident mice, squirrels, and chipmunks. Overall, we found little evidence of competition. There were essentially no within-season numerical responses to changes in the abundance of putative competitors. Moreover, while individual-level responses (apparent survival and capture probability) did vary with competitor densities in some models, these effects were often better explained by site-specific parameters and were restricted to few of the 19 sites we studied. With only weak or nonexistent competition among these three common rodent species, we expect their patterns of habitat occupancy and population dynamics to be largely independent of one another.     

Identifying Darwinian Selection Acting on Different Human APOL1 Variants among Diverse African Populations

Disease susceptibility can arise as a consequence of adaptation to infectious disease. Recent findings have suggested that higher rates of chronic kidney disease (CKD) in individuals with recent African ancestry might be attributed to two risk alleles (G1 and G2) at the serum-resistance-associated (SRA)-interacting-domain-encoding region of APOL1. These two alleles appear to have arisen adaptively, possibly as a result of their protective effects against human African trypanosomiasis (HAT), or African sleeping sickness. In order to explore the distribution of potential functional variation at APOL1, we studied nucleotide variation in 187 individuals across ten geographically and genetically diverse African ethnic groups with exposure to two Trypanosoma brucei subspecies that cause HAT. We observed unusually high levels of nonsynonymous polymorphism in the regions encoding the functional domains that are required for lysing parasites. Whereas allele frequencies of G2 were similar across all populations (3%–8%), the G1 allele was only common in the Yoruba (39%). Additionally, we identified a haplotype (termed G3) that contains a nonsynonymous change at the membrane-addressing-domain-encoding region of APOL1 and is present in all populations except for the Yoruba. Analyses of long-range patterns of linkage disequilibrium indicate evidence of recent selection acting on the G3 haplotype in Fulani from Cameroon. Our results indicate that the G1 and G2 variants in APOL1 are geographically restricted and that there might be other functional variants that could play a role in HAT resistance and CKD risk in African populations.     

Separation of Assembly-Competent Tubulin from Brain Microtubule Protein Preparations Using a Fast-Performance Liquid Chromatography Procedure

Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. Microtubule protein (1-100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KCl with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., less than 1 versus 60 mU/mg/min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmol/mg/min of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotide/tubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37 degrees C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of fire on bird diversity and abundance in an East African savanna

Fire is an important determinant of many aspects of savanna ecosystem structure and function. However, relatively little is known about the effects of fire on faunal biodiversity in savannas. We conducted a short‐term study to examine the effects of a replicated experimental burn on bird diversity and abundance in savanna habitat of central Kenya. Twenty‐two months after the burn, Shannon diversity of birds was 32% higher on plots that had been burned compared with paired control plots. We observed no significant effects of burning on total bird abundance or species richness. Several families of birds were found only on plots that had been burned; one species, the rattling cisticola (Cisticola chiniana), was found only on unburned plots. Shrub canopy area was negatively correlated with bird diversity on each plot, and highly correlated with grass height and the abundance of orthopterans. Our results suggest that the highest landscape‐level bird diversity might be obtained through a mosaic of burned and unburned patches. This is also most likely to approximate the historical state of bird diversity in this habitat, because patchy fires have been an important natural disturbance in tropical ecosystems for millennia.     

Enzyme-linked enzyme-binding assay for Pin1 WW domain ligands

Peptidyl prolyl cis-trans isomerase (PPIase) interacting with NIMA-1 (Pin1) catalyzes the cis-trans isomerization of pSer/pThr-Pro amide bonds. Pin1 is a two-domain protein that represents a promising target for the treatment of cancer. Both domains of Pin1 bind the pSer/pThr-Pro motif; PPIase enzymatic activity occurs in the catalytic domain, and the WW domain acts as a recognition module for the pSer/pThr-Pro motif. An assay we call an enzyme-linked enzyme-binding assay (ELEBA) was developed to measure the K_d of ligands that bind selectively to the WW domain. A ligand specific for the WW domain of Pin1 was covalently immobilized in a 96-well plate. Commercially available Pin1 conjugated to horseradish peroxidase was used for chemiluminescent detection of ligands that block the association of the WW domain with immobilized ligand. The peptide ligands were derived from the cell cycle regulatory phosphatase, Cdc25c, residues 45-50. The K_d values for Fmoc-VPRpTPVGGGK-NH₂ and Ac-VPRpTPV-NH₂ were determined to be 36 ± 4 and 110 ± 30 μM, respectively. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain, even in the presence of the catalytic domain. This method may be applied to any dual specificity, multidomain protein.     

Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.