High affinity binding of Hsp90 is triggered by multiple discrete segments of its kinase clients

The 90 kDa heat shock protein (Hsp90) cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, crystal structures of protein kinases were used to guide the dissection of two kinases [the Src-family tyrosine kinase, Lck, and the heme-regulated eIF2alpha kinase (HRI)], and the association of Hsp90 and Cdc37 with these constructs was assessed. Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the kinases' catalytic domains. In contrast, Cdc37 interacted only with the NL. The Hsp90 antagonist molybdate was necessary to stabilize the interactions between isolated subdomains and Hsp90 or Cdc37, but the presence of both lobes of the kinases' catalytic domain generated a stable salt-resistant chaperone-client heterocomplex. The Hsp90 co-chaperones FKBP52 and p23 interacted with the catalytic domain and the NL of Lck, whereas protein phosphatase 5 demonstrated unique modes of kinase binding. Cyp40 was a salt labile component of Hsp90 complexes formed with the full-length, catalytic domains, and N-terminal catalytic lobes of Lck and HRI. Additionally, dissections identify a specific kinase motif that triggers Hsp90's conformational switching to a high-affinity client binding state. Results indicate that the Hsp90 machine acts as a versatile chaperone that recognizes multiple regions of non-native proteins, while Cdc37 binds to a more specific kinase segment, and that concomitant recognition of multiple client segments is communicated to generate or stabilize high-affinity chaperone-client heterocomplexes.     

Antagonistic effect of fluorescent pseudomonads against Macrophomina phaseolina that causes charcoal rot of groundnut

Maximum colony growth inhibition was observed due to Pseudomonas PS2 (74%) as compared to PS1 (71%) on trypticase soy agar (TSM) plates after 5 days of incubation. Light and scanning electron microscopic examination showed hyphal coiling, vacuolation, coagulation and granulation of cytoplasm resulting in lysis of hyphae of M. phaseolina by pseudomonads. Cell free culture filtrates of strains PS1 and PS2 restricted the growth of mycelium of M. phaseolina. PS1 and PS2 caused maximum colony growth inhibition by 57 and 61% respectively at 20% concentration of culture filtrate after 4 days of incubation. Volatile substances produced by PS1 and PS2 also inhibited the colony growth of M. phaseolina by 25 and 32%, respectively. Inhibitory effect of volatile substances, however, decreased with advancing in incubation period. Colony growth of M. phaseolina was significantly decreased by PS1 and PS2 as compared to control both in iron- sufficient and iron-deficient conditions. PS2 showed higher antagonistic activity than PS1, as evidenced by pronounced colony growth inhibition.     

Anchorage of oligonucleotide hybridization by tethered phenazine nucleoside analogue

UV absorption and fluorescence techniques with a thermal denaturation procedure were used in studies of the anchorage of an oligonucleotide hybridization by a covalently tethered nucleoside analogue of an intercalating imidazophenazine derivative (Pzn). The formation by the (dT)(10)Pzn conjugate of the duplex complex with (dA)(15) and the triplex complex with (dA)(15) or poly(dA).poly(dT) was studied in buffered solutions with 0.11 and/or 1M sodium ions at the oligomer strand concentration of 10 microM. Because of the Pzn emission sensitivity to the interaction with adenine bases, a fluorescence technique was found to be effective in the detection of melting transitions. The attached Pzn substantially enhanced the thermal stability of complexes formed by (dT)(10) because of the intercalation mechanism, which increased the temperature of half-dissociation of the duplex by 10-12 degrees C and of the triplexes by approximately 13 degrees C. With the assumption of a two-state model of transition, the thermodynamic parameters for duplex formations were derived. The investigated variant of conjugation has a certain advantage over the widely used attachment via a flexible linker, consisting of a predetermined location of the Pzn chromophore in target sequences that makes it useful as a fluorescent reporter of the hybridization correctness. Molecular modeling was used to construct the geometries of the intercalation sites that turned out to be in conformity with the behavior of the Pzn fluorescence.     

IL-8 directly enhanced endothelial cell survival,proliferation, and matrix metalloproteinases production and regulated angiogenesis

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-x(L):Bcl-x(S) and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis.     

Toxoplasma gondii isolates from free-ranging chickens from the United States

Prevalence of Toxoplasma gondii infection in chickens is a good indicator of the strains prevalent in their environment because they feed from ground. The prevalence of T. gondii was determined in 118 free-range chickens from 14 counties in Ohio and in 11 chickens from a pig farm in Massachusetts. Toxoplasma gondii antibodies (> or = 1: 5) were found using the modified agglutination test (MAT) in 20 of 118 chickens from Ohio. Viable T. gondii was recovered from 11 of 20 seropositive chickens by bioassay of their hearts and brains into mice. The parasite was not isolated from tissues of 63 seronegative (< or = 1:5) chickens by bioassay in cats. Hearts, brains, and muscles from legs and breast of the 11 chickens from the pig farm in Massachusetts were fed each to a T. gondii-negative cat. Eight cats fed chicken tissues shed oocysts; the 3 cats that did not shed oocysts were fed tissues of chickens with MAT titers of 1:5 or less. Tachyzoites of 19 isolates of T. gondii from Ohio and Massachusetts were considered avirulent for mice. Of 19 isolates genotyped, 5 isolates were type II and 14 were type III; mixed types and type I isolates were not found.     

Immunization of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protection against vertical transmission of Neospora caninum

The purpose of the present study was to use direct plasmid deoxyribonucleic acid (DNA) injection to identify specific antigens that confer protection against congenital transfer of Neospora caninum. Inbred BALB/c mice were vaccinated before pregnancy with a recombinant plasmid containing sequences encoding N. caninum antigen NcGRA7 or NcsHSP33. The mice were challenged with N. caninum tachyzoites at 10-12 days of gestation. Whereas 100% of pups born from dams immunized with control plasmid contained detectable levels of N. caninum DNA in a Neospora-specific polymerase chain reaction assay, only 46% of pups from pCMVi-NcGRA7-immunized mice and 53% of pCMVi-NcsHSP33-immunized mice were N. caninum positive, and none of the mice immunized with tachyzoite extract contained N. caninum DNA. Thus, immunization of mice with plasmid DNA expressing N. caninum antigens conferred partial protection against congenital neosporosis.     

An esterase is involved in geraniol production during palmarosa inflorescence development

Total incorporation of exogenously administered [2-14C]acetate into essential oil of palmarosa (Cymbopogon martinii) was found to be relatively higher than that of either [U-14C]sucrose or [U-14C]glucose during inflorescence development. Among the major essential oil constituents, biogenesis of geranyl acetate was much higher than that of geraniol. Alkaline hydrolysis of [14C]labeled geranyl acetate revealed that the majority of the label incorporated into geranyl acetate was present in the geraniol moiety, indicating that only newly synthesized geraniol gets acetylated to form geranyl acetate. Geranyl acetate cleaving esterase (GAE) activity followed a similar pattern during both in vivo and in vitro inflorescence development, with maximum activity at immature inflorescence stages, suggesting the involvement of GAE in geraniol production during inflorescence development. Five esterase isozymes (Est-A to E) were detected in the enzymic fraction of palmarosa inflorescence and all showed GAE activity, with Est-B being significantly increased during inflorescence development. The role of GAE in geraniol production and improving the palmarosa oil quality is discussed.     

Photoaptamer arrays applied to multiplexed proteomic analysis

Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.     

Zoonotic protozoa: from land to sea

Attention to worldwide pollution of the coastal marine environment has focused primarily on toxic algal blooms and pathogenic bacteria that multiply in nutrient-rich waters. However, massive but unseen amounts of feces from humans, their pets, and their domesticated animals are discharged, dumped, or carried in runoff, bringing encysted zoonotic protozoan parasites to estuaries and coastal waters. Here, they contaminate bathing beaches, are filtered and concentrated by shellfish eaten by humans and marine mammals, and infect a wide range of marine animal hosts, resulting in morbidity and mortality to some populations. This review addresses the extent of contamination and the animals affected by three genera of important zoonotic protozoa: Giardia, Cryptosporidium and Toxoplasma.     

Biological and molecular characterization of Besnoitia akodoni n.sp. (Protozoa: Apicomplexa) from the rodent Akodon montensis in Brazil

The diversity among coccidian parasites of the genus Besnoitia is incompletely known. Of the eight currently described members of the genus, only B. jellisoni is known to parasitize a rodent host. Here, we propose a new name, Besnoitia akodoni, for the species initially isolated form the rodent Akodon montensis in Brazil. The tissue cysts of B. akodoni were up to 442 microm in diameter and bradyzoites were 8.4 x 1.4 microm in size. The bradyzoites contained enigmatic bodies, micronemes and rhoptries. Tachyzoites were 5.8 x 1.5 microm in size and they could be grown in vitro in bovine monocytes and African Green monkey cells where they divided by endodyogeny. Besnoitia akodoni was infective to laboratory-raised mice (Mus musculus) and gerbils (Meriones unguiculatus) but not to cats (Felis catus). Comparison of the conserved sequences of the small subunit rDNA clearly established the close relationship of B. akodoni with other members of the genus. However, sequences of the more variable first internal transcribed spacer portion of the ribosomal DNA repeat support its differentiation from the other species of the genus.