Parasitism and decreased response to sex pheromones in malePeriplaneta americana (Dictyoptera: Blattidae)

Parasites are known to alter the behavior of their hosts, but little is known about their effects on responses to sexual stimuli. Periplaneta americanainfected with the acanthocephalan Moniliformis moniliformiswere compared to uninfected animals in their behavioral and electroantennogram responses to a synthetic P. americanapheromone component, periplanone-B, and a pheromone mimic, bornyl acetate. In a t-maze there was no significant difference between infected animals' responses to periplanone-B and a random binomial distribution; the responses of uninfected animals were significantly nonrandom. The electroan-tennogram responses of infected and uninfected animals to bornyl acetate or periplanone-B did not differ significantly, however, indicating that the alteration probably does not occur at the peripheral level but at a central nervous system level.     

Orally active and brain permeable proline amides as highly selective 5HT2c agonists for the treatment of obesity

Brain-penetrable proline amides were developed as 5HT2c agonists with more than 1000-fold binding selectivity against 5HT2b receptor. After medicinal chemistry optimization and SAR studies, orally active proline amides with robust efficacy in a rodent food intake inhibition model were uncovered.     

Rootstock genotype succession influences apple replant disease and root-zone microbial community composition in an orchard soil

Apple replant disease (ARD) is a soil-borne disease complex that affects young apple trees in replanted orchards, resulting in stunted growth and reduced yields. Newly developed rootstock genotypes with tolerance to ARD may help to control this disease. We determined the effects of rootstock genotype rotations during orchard renovation, by investigating root-zone soil microbial consortia and the relative severity of ARD on seven rootstock genotypes (M.9, M.26, G.30, G.41, G.65, G.935, and CG.6210) planted in soil where trees on four of those same rootstocks (M.9, M.26, G.30 and CG.6210) had grown for the previous 15 years. Rootstock genotyping indicated that genetic distances among rootstocks were loosely correlated with their differential responses to ARD. Root-zone fungal and bacterial community composition, assessed by DNA fingerprinting (T-RFLP), differed between M.26 and CG.6210. Soil bacterial communities were influenced most by which rootstock had grown in the soil previously, while fungal communities were influenced more by the current replanted rootstock. In a clone library of bacteria from M.26 and CG.6210 root-zone soil, β-Proteobacteria was the most abundant phylum (25% of sequences). Sequences representing the Burkholderia cepacia complex were obtained only from CG.6210 soil. Rootstock genotypes that were grown in the orchard soil previously affected subsequent ARD severity, but replanting with the same or closely related rootstocks did not necessarily exacerbate this disease problem. Our results suggest that genotype-specific interactions with soil microbial consortia are linked with apple rootstock tolerance or susceptibility to ARD.     

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.     

Concentrations,Viability, and Distribution of Cryptosporidium Genotypes in Lagoons of Swine Facilities in the Southern Piedmont and in Coastal Plain Watersheds of Georgia

Waste lagoons of swine operations are a source of Cryptosporidium oocysts. Few studies, however, have reported on oocyst concentrations in swine waste lagoons; none have reported on oocyst viability status, nor has there been a systematic assessment of species/genotype distributions across different types of swine facilities. Ten swine waste lagoons associated with farrowing, nursery, finishing, and gestation operations were each sampled once a month for a year. Oocysts were extracted from triplicate 900-ml effluent samples, enumerated by microscopy, and assessed for viability by dye exclusion/vital stain assay. DNA was extracted from processed samples, and 18S ribosomal DNA (rDNA) genes were amplified by PCR and sequenced for species and genotype identification. Oocysts were observed at each sampling time at each lagoon. Annual mean concentrations of total oocysts and viable oocysts ranged between 24 and 51 and between 0.6 and 12 oocysts ml⁻¹ effluent, respectively. The species and genotype distributions were dominated (95 to 100%) by Cryptosporidium suis and Cryptosporidium pig genotype II, the latter of which was found at eight of the lagoons. The lagoon at the gestation facility was dominated by Cryptosporidium muris (90%), and one farrowing facility showed a mix of pig genotypes, Cryptosporidium muris, and various genotypes of C. parvum. The zoonotic C. parvum bovine genotype was observed five times out of 407 18S rDNA sequences analyzed. Our results indicate that pigs can have mixed Cryptosporidium infections, but infection with C. suis is likely to be dominant.Over the last few decades, pork production in North America has undergone significant growth and centralization into large concentrated swine (Sus scrofa) operations with more animals on fewer farms (18). A consequence of the increase in numbers of swine per facility is a concomitant increased concentration of swine waste. Present housing facilities for swine are designed to collect feces and urine in wastewater lagoons, in which the waste undergoes anaerobic transformations. One of several public health concerns over swine lagoons is the potential presence of infectious bacteria, viruses, and protozoa (4). Because of the notoriety given to swine waste lagoon spills in the coastal flood plain of North Carolina that were associated with a series of hurricanes in 1998 and 1999 (21), large-scale swine operations have become a focus of environmental and public health concerns.The cause of the massive outbreak of cryptosporidiosis in Milwaukee, WI, in 1993 was afterwards determined to be Cryptosporidium hominis, the human genotype of C. parvum and an obligate parasite of humans (33, 44). At the time, however, it was thought to be caused by C. parvum (22). Because of this initial misidentification of the cryptosporidial source of the outbreak, the connection between C. parvum and large-scale confined livestock operations has become a focused area of research. Although manure-associated outbreaks of C. parvum have implicated bovine sources, a Canadian study found that the prevalence of Cryptosporidium in swine lagoons was greater than that in dairy liquid manure (9). Olson et al. (24) also reported the presence of Cryptosporidium oocysts of undetermined genotype at four of six hog operations in Canada. Atwill et al. (2) observed C. parvum oocysts in feces of feral pigs. Hutchison et al. (13) observed C. parvum oocysts of undetermined genotype in 5 and 13% of fresh and stored fecal samples, respectively, from pigs of undeclared age. Guselle et al. (10) followed the course of a naturally occurring C. parvum infection in 33 weaned pigs. Following the protocol of the genetic analysis of Morgan et al. (23), Guselle et al. (10) identified this C. parvum genotype as being adapted to pigs. At the time, the zoonotic potential of this C. parvum pig-adapted genotype was considered uncertain (23).Recently, two genotypes of Cryptosporidium have been recognized as host adapted to swine: Cryptosporidium suis (formerly Cryptosporidium pig genotype I) and Cryptosporidium pig genotype II (28, 29). Xiao et al. (37) reported on an immunocompromised person who was infected with a Cryptosporidium pig genotype and thus implicated Cryptosporidium from swine as potentially zoonotic and a public health concern. Before molecular methods were developed to differentiate pig genotypes of Cryptosporidium from other species, C. parvum was thought to infect 152 species of mammals and consist of several cryptic species (6). An extensive survey of swine effluent from swine finishing operations in Ireland indicated a prevalence of both C. suis and Cryptosporidium pig genotype II (39). Hamnes et al. (11) reported prevalence of both C. suis and Cryptosporidium pig genotype II in feces of suckling pigs across Norway and thus implicated farrowing operations as sources of this parasite.Other than the prevalence of Cryptosporidium in feces of young pigs and effluent lagoons of older pigs in finishing operations, little comprehensive data on oocyst concentrations, viability of oocysts, and distributions of Cryptosporidium species and genotypes have been reported. No systematic study of swine lagoon effluents from large-scale facilities has been reported for the four separate stages of swine development, (i) breeding and gestation, (ii) farrowing (parturition), (iii) nursery (in which weaned piglets are kept until 8 to 9 weeks of age), and (iv) finishing (in which 8- to 9-week-old pigs are kept to market weight). The objective of this investigation was to determine for 1 year the frequencies, concentrations, viability statuses, and distributions of Cryptosporidium species and genotypes in lagoons associated with the four types of swine operations in the Southern Piedmont and in coastal plain watersheds of Georgia.     

Screening Microalgae Strains for Biodiesel Production: Lipid Productivity and Estimation of Fuel Quality Based on Fatty Acids Profiles as Selective Criteria

The viability of algae-based biodiesel industry depends on the selection of adequate strains in regard to profitable yields and oil quality. This work aimed to bioprospecting and screening 12 microalgae strains by applying, as selective criteria, the volumetric lipid productivity and the fatty acid profiles, used for estimating the biodiesel fuel properties. Volumetric lipid productivity varied among strains from 22.61 to 204.91 mg l^?1 day^?1. The highest lipid yields were observed for Chlorella (204.91 mg l^?1 day¹) and Botryococcus strains (112.43 and 98.00 mg l^?1 day^?1 for Botryococcus braunii and Botryococcus terribilis, respectively). Cluster and principal components analysis analysis applied to fatty acid methyl esters (FAME) profiles discriminated three different microalgae groups according to their potential for biodiesel production. Kirchneriella lunaris, Ankistrodesmus fusiformis, Chlamydocapsa bacillus, and Ankistrodesmus falcatus showed the highest levels of polyunsaturated FAME, which incurs in the production of biodiesels with the lowest (42.47–50.52) cetane number (CN), the highest (101.33–136.97) iodine values (IV), and the lowest oxidation stability. The higher levels of saturated FAME in the oils of Chlamydomonas sp. and Scenedesmus obliquus indicated them as source of biodiesel with higher oxidation stability, higher CN (63.63–64.94), and lower IV (27.34–35.28). The third group, except for the Trebouxyophyceae strains that appeared in isolation, are composed by microalgae that generate biodiesel of intermediate values for CN, IV, and oxidation stability, related to their levels of saturated and monosaturated lipids. Thus, in this research, FAME profiling suggested that the best approach for generating a microalgae-biodiesel of top quality is by mixing the oils of distinct cell cultures.     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Functional analysis of an auxin-inducible DNA-binding protein gene

Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin–induced callus formation and reduced apical dominance.     

Invariant chain modulates HLA class II protein recycling and peptide presentation in nonprofessional antigen presenting cells

The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecules access antigenic peptides by distinct, but poorly defined pathways in the absence of Ii. Here, investigations demonstrate that nonprofessional APC such as human fibroblasts lacking Ii internalize antigenic peptides prior to the binding of these ligands to recycling class II molecules. By contrast, fibroblast lines expressing Ii favor exogenous peptides binding directly to cell surface class II molecules without a need for ligand internalization. Endocytosis of class II molecules was enhanced in cells lacking Ii compared with Ii-expressing APC. These results suggest enhanced reliance on the endocytic recycling pathway for functional class II presentation in nonprofessional APC.