Seroepidemiology of infection with Toxoplasma gondii in workers occupationally exposed to water, sewage, and soil in Durango, Mexico

Water, sewage, and soil are potential sources of infection for Toxoplasma gondii. Therefore, we sought to determine the prevalence of T. gondii infection and associated characteristics in 61 plumbers, 203 construction workers, and 168 gardeners in Durango City, Mexico. Participants were tested for T. gondii IgG and IgM antibodies with the use of enzyme-linked immunoassays. In addition, sociodemographic, clinical, and behavioral characteristics from each participant were obtained. IgG T. gondii antibodies were found in 4 (6.6%) plumbers, 17 (8.4%) construction workers, and 10 (6.0%) gardeners; T. gondii IgM antibodies were found in 3 (1.5%) construction workers and 4 (2.4%) gardeners, but in none of the plumbers. In the total population, the prevalence of T. gondii infection was significantly higher in workers living in suburban areas, without education, workers that consumed chorizo, and those who suffered from any disease (P < 0.05). In gardeners, prevalence of infection was significantly higher in those with blood transfusion, and memory impairment (P < 0.05). Multivariate analysis showed that T. gondii infection was positively associated with consumption of unwashed fruits (adjusted odds ratio [OR] = 2.41; 95% confidence interval [CI]: 1.13-5.13), and with raising animals (adjusted OR = 2.53; 95% CI: 1.00-6.37). This is the first report of contributing factors for T. gondii infection in workers occupationally exposed to water, sewage, and soil in a Mexican city, and results will contribute to the design of optimal preventive measures.     

Serological and parasitological prevalence of Toxoplasma gondii in wild birds from Colorado

Ground-feeding birds are considered important in the epidemiology of Toxoplasma gondii because they serve as indicators of soil contamination by oocysts, and birds of prey are indicators of T. gondii prevalence in rodents and other small mammals. Cats excrete environmentally resistant oocysts after consuming tissues of T. gondii -infected birds. In the present study, sera and tissues from 382 wild birds from Colorado were tested for T. gondii infection. Antibodies to T. gondii were found in 38 birds with the use of the modified agglutination test (MAT, 1∶25 titer). Tissues (brains, hearts) of 84 birds were bioassayed in mice. Viable T. gondii was isolated from 1 of 1 barn owl (Tyto alba), 1 of 5 American kestrels (Falco sparverius), 1 of 7 ferruginous hawks (Buteo regalis), 1 of 4 rough-legged hawks (Buteo lagopus), 2 of 13 Swainson's hawks (Buteo swainsoni), and 1 of 25 red-tailed hawks (Buteo jamaicensis). This is the first time T. gondii has been isolated from the barn owl, ferruginous hawk, rough-legged hawk, and Swainson's hawk.     

Waterborne toxoplasmosis - Recent developments

Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the USA provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water.     

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.     

Conversion of type I 4:6 to 3:5 beta-turn types in human acidic fibroblast growth factor: effects upon structure, stability, folding, and mitogenic function

Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 contains 11 beta-turns, the majority being type I 3:5; however, a type I 4:6 turn is also found at three symmetry-related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of deletion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional sequence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local environment). Although the results suggest that a stable, foldable beta-trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn 1 of FGF-1 appears essential for efficient mitogenic function.     

Snapshots of protein folding problem: implications of folding and misfolding studies

Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed.     

Modulation of serum cortisol by substance P in albino rats: evidence of a direct effect on adrenal gland

Effect of prolonged administration of substance P on the plasma cortisol level in the albino rats has been investigated. An inhibitory impact on intact individuals and a stimulatory effect in pharmacologically annulled rats has been observed. It is concluded that in normal conditions substance P presumably acts as a preventive agent for any excess secretion of cortisol while during stress or disturbed HPA or RAS conditions, it stimulates the secretion of cortisol. An intraglandular modulatory role of substance P has been suggested.     

A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K(m) of 7.57 mM and V(max) of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 degrees C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.     

Mechanistic insights into the dual inhibition strategy for checking Leishmaniasis

Leishmaniasis (1) is an endemic disease mainly caused by the protozoan Leishmania donovani (Ld). Polyamines have been identified as essential organic compounds for the growth and survival of Ld. These are synthesized in Ld by polyamine synthesis pathway comprising of many enzymes such as ornithine decarboxylase (ODC), spermidine synthase (SS), and S-adenosylmethionine decarboxylase. Inhibition of these enzymes in Ld offers a viable prospect to check its growth and development. In the present work, we used computational approaches to search natural inhibitors against ODC and SS enzymes. We predicted three-dimensional structures of ODC and SS using comparative modeling and molecular dynamics (MD) simulations. Thousands of natural compounds were virtually screened against target proteins using high throughput approach. MD simulations were then performed to examine molecular interactions between the screened compounds and functional residues of the active sites of the enzymes. Herein, we report two natural compounds of dual inhibitory nature active against the two crucial enzymes of polyamine pathway of Ld. These dual inhibitors have the potential to evolve as lead molecules in the development of antileishmanial drugs. (1)These authors contributed equally.     

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.