Elevated genetic diversity of mitochondrial genes in asexual populations of Bark Lice ('Psocoptera': Echmepteryx hageni)

Asexual reproduction is commonly thought to be associated with low genetic diversity in animals. Echmepteryx hageni (Insecta: 'Psocoptera') is one of several psocopteran species that are primarily parthenogenetic, but also exists in small, isolated sexual populations. We used mitochondrial DNA sequences to investigate the population history and genealogical relationships between the sexual and asexual forms of this species. The asexual population of E. hageni exhibits extremely high mitochondrial haplotype diversity (H=0.98), whereas the sexual forms had significantly lower haplotypic diversity (H=0.25, after correcting for sample size). This diversity in asexuals represents one the greatest genetic diversities reported for asexual animals in the literature. Nucleotide diversities were also higher in asexual compared to sexual populations (π=0.0071 vs. 0.00027). Compared to other reported estimates of π in insects, asexual nucleotide diversity is high, but not remarkably elevated. Three hypotheses might explain the elevated genetic diversity of asexual populations: (i) larger effective population size, (ii) greater mutation rate or (iii) possible recent origin of sexuals. In addition, phylogeographic analysis revealed little geographic structure among asexual E. hageni, although specimens from the upper Midwest form a single clade and are genetically differentiated. The mismatch distribution and neutrality tests indicate a historical population size increase, possibly associated with expansion from glacial refugia.     

Binding and cleavage of unstructured RNA by nuclear RNase P

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.     

The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the Hip1-related protein Sla2 delayed the formation of extended plasma membrane invaginations in sjl2ts cells, indicating that Sla2 may become limiting or misregulated in cells with impaired PI phosphatase activity. Consistent with this, the cortical actin patch protein Sla2 is mislocalized in sjl1Delta sjl2Delta mutant cells. Together, our studies suggest that PI metabolism by the synaptojanin-like proteins coordinately directs actin dynamics and membrane invagination, in part by regulation of Sla2.     

The molecular basis of odor coding in the Drosophila larva

We have analyzed the molecular basis of odor coding in the Drosophila larva. A subset of Or genes is found to be expressed in larval olfactory receptor neurons (ORNs). Using an in vivo expression system and electrophysiology, we demonstrate that these genes encode functional odor receptors and determine their response spectra with 27 odors. The receptors vary in their breadth of tuning, exhibit both excitation and inhibition, and show different onset and termination kinetics. An individual receptor appears to transmit signals via a single ORN to a single glomerulus in the larval antennal lobe. We provide a spatial map of odor information in the larval brain and find that ORNs with related functional specificity map to related spatial positions. The results show how one family of receptors underlies odor coding in two markedly different olfactory systems; they also provide a molecular mechanism to explain longstanding observations of larval odor discrimination.     

Purification and stabilization of ricin B from tobacco hairy root culture medium by aqueous two-phase extraction

Ricin B (RTB), the non-toxic lectin subunit of ricin, is a promising mucosal adjuvant and carrier for use in humans. RTB fusion proteins have been expressed in tobacco hairy root cultures, but the secreted RTB component of these proteins was vulnerable to protease degradation in the medium. Moreover, castor bean purified RTB spiked into tobacco hairy root culture media showed significant degradation after 24 h and complete loss of product after 72 h. Aqueous two-phase extraction (ATPE) was tested for fast recovery of RTB not only to partially purify the protein but also to improve its stability. Two different polyethylene glycol (PEG)/salt/water systems including PEG/potassium phosphate and PEG/sodium sulfate, were studied. RTB was shown to be favorably recovered in PEG/sodium sulfate systems. Statistical analysis indicated that the ionic strength of the system and the sodium sulfate concentration were important in optimizing the partition coefficient of RTB. A selectivity of almost three could be achieved for RTB in optimized systems, and RTB partitioned in the PEG-rich phase exhibited extended stability. Therefore, ATPE was shown to be effective in initial recovery/purification and stabilization of RTB and may hold promise for other unstable secreted proteins from hairy root culture.     

Effect of soil potassium availability on soybean aphid (Hemiptera: Aphididae) population dynamics and soybean yield

Studies were conducted to examine the effect of potassium (K) on soybean aphid, Aphis glycines Matsumura, population growth. A laboratory feeding assay examined the effect of K-deficient foliage on life table parameters of soybean aphids, and field experiments were designed to determine the effect of three soil K treatment levels on aphid populations and their impact on soybean yields. The feeding assay found that life table parameters differed between aphids feeding on the K-deficient and nondeficient soybean leaves. Soybean aphids in the K-deficient treatment exhibited significantly greater intrinsic rate of increase (r(m)), finite rate of increase (lambda), and net reproductive rate (Ro) relative to aphids feeding on nondeficient leaves. No significant difference was observed in mean generation time (T) between the two treatments. However, the field experiment repeated over 2 yr showed no effect of K on soybean aphid populations. Soybean aphid populations were high in unsprayed plots and feeding resulted in significant yield losses in 2002 at all three K treatment levels: when averaged across 2001 and 2002, unsprayed treatments yielded 22, 18, and 19.5% less than the sprayed plots in the low, medium, and high K treatments, respectively. No significant interaction was observed between aphid abundance and K level on soybean yields in either year. This study therefore suggests that although aphids can perform better on K-deficient plants, aphid abundance in the field may be dependent on additional factors, such as dispersal, that may affect final densities within plots.     

Crystal structures of human HSP90alpha-complexed with dihydroxyphenylpyrazoles

A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90alpha were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.     

The structure of an antitumor C(H)2-domain-deleted humanized antibody

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.     

Microbial fuel cells meet with external resistance

The influence of external load on the composition of the anodic biofilm microbial community and biomass yield was investigated in a microbial fuel cell fed with glucose and domestic wastewater was used as source of electrogens. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments revealed distinct differences in anodic bacterial communities formed at the anode of each MFC operated under a different external load. These results implied that in an MFC, electrogenic bacteria were enriched under higher current densities, i.e., low external load, and were able to sustain better current and effluent quality. The influence of the external resistance applied to the MFCs during formation of the bacterial communities from sewage wastewater was shown to have no significant effect on power performance of the MFCs nor to have a significant influence on their anodic activity with both glucose and brewery wastewater as fuel. As expected, current generation, COD removal and the biomass yield were all directly influenced by the external load. Significantly, when operated under lower external load, the biomass yield in the MFC was less than that in conventional anaerobic digestion (i.e., control).     

In vitro and in vivo evaluation of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors

Synthesis and biological evaluation of a series of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and pharmacodynamic effects.