Bioaerosols from municipal and animal wastes: background and contemporary issues

Global population increases, coupled with intensive animal and livestock production practices, have resulted in the generation, accumulation, and disposal of large amounts of wastes around the world. Aerosolization of microbial pathogens, endotoxins, odors, and dust particles is an inevitable consequence of the generation and handling of waste material. Bioaerosols can be a source of microbial pathogens, endotoxins, and other allergens. Given the close proximity of population centers to concentrated animal-rearing operations and municipal treatment facilities in many parts of the world, there is concern regarding the occupational and public health impacts associated with the exposure to bioaerosols from municipal and animal wastes. Major advances have been made in our understanding of bioaerosol characteristics, identifying the hazards, and identifying possible human and animal health links with aerosolized pathogens and allergens. However, significant knowledge and technology gaps still exist. These include a lack of clear understanding of the fate and transport of bioaerosols, especially within the open environment, an inability to accurately predict the health risks associated with bioaerosolized pathogens, and a lack of standardized bioaerosol sampling protocols, and efficient samplers. This review synthesizes the information related to bioaerosols and addresses the contemporary issues associated with bioaerosols from municipal and animal wastes, with a focus on pathogens.     

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.     

Stereoselective synthesis of a C-glycosylic compound (a "methyl C-glycoside") through a regioselective free-radical ring-opening reaction. A single-crystal X-ray structure determination

Readily available 3,4,6-tri-O-acetyl-D-glucal was converted to 2,6-anhydro-5,7-O-benzylidene-1,3,4-trideoxy-D-arabino-hept-3-enitol, a methyl C-glycosylic compound. Cyclopropanation of 4,6-O-benzylidene-D-glucal, followed by tributylstannyl radical-mediated regioselective ring opening of the 1,2-cyclopropano sugar led to a 2,6-anhydro-1-deoxyheptose, (a "methyl C-beta-D-glycoside"). The stereochemistry of the 1,2-cyclopropano sugar and the "methyl C-glycoside" were confirmed by single-crystal X-ray diffraction studies.     

Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 g) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 ± 0.2g/mg total protein) was 10 times that in normal tissue (0.1 ±0.01 g/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 ± 0.3g/mg) was 15 times that in normal tissue (0.12 ± 0.02 g/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.     

Muscle fibre growth dynamics in diploid and triploid rainbow trout

The effect of triploidy on muscle fibre growth was determined by comparing hyperplasia and hypertrophy of white muscle fibres in all-female, diploid and triploid rainbow trout Oncorhynchus mykiss (100–400 mm total length). Conventional morphometry and protein and DNA concentrations were used to assess muscle fibre hyperplasia and hypertrophy in white muscle samples derived from an anterio-dorsal location. Muscle fibre distributions were significantly different between triploids and diploids in trout <300 mm. The proportion of fibres <20 μm was higher in diploids than in triploids and the proportion of fibres in the 20–40 μm category was higher in triploids than in diploids. This indicates that the hyperplastic fibres of triploids are larger than those of diploids. Larger hyperplastic fibres in triploids are probably due to the combined effect of increased nuclear size in triploids and the relatively high nucleus: cell ratio observed in small muscle fibres. These larger fibres may be less favourable to cellular metabolic exchange because of their smaller surface area to volume ratios, and perhaps account for reduced viability and growth observed in triploids during early life stages. On the other hand, the lack of difference in the distribution of fibres <20 μm between diploids and triploids at larger body size ranges (301–400 mm) imply that triploid trout may have higher rates of new fibre recruitment and growth capacity at these sizes. There was no difference between diploid and triploid trout in the mean size of muscle fibres; however, the number of fibres per unit area was reduced by 10% in triploids. No differences were observed in protein or DNA concentrations in muscle tissues between the two genetic groups. Since triploid nuclei have 1·5 times more DNA than diploid nuclei, this deviation from the expected muscle DNA concentration (1·3–1·4 times more DNA in triploids when the 10% reduction in fibre density is considered) suggests that the number of nuclei per muscle fibre is reduced. In both diploids and triploids, mean fibre size increased with body length while fibre density decreased. Similarly, protein concentration in the muscle tissue increased and DNA concentration declined with increasing body length. Protein/DNA ratio was strongly and positively correlated with fibre size. These results demonstrate that changes in DNA and protein concentrations can be used to assess hyperplasia and hypertrophy in muscle tissues. However, the morphometric procedure provides better insight into muscle fibre growth as it enables the direct visualization and analysis of muscle fibre distribution patterns.     

Products of Proline Catabolism Can Induce Osmotically Regulated Genes in Rice

Many plants accumulate high levels of free proline (Pro) in response to osmotic stress. This imino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The present study provides evidence that the synthesis of Pro may have an additional effect. We found that intermediates in Pro biosynthesis and catabolism such as glutamine and Δ¹-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated genes in rice (Oryza sativa L.), including salT and dhn4. One millimolar P5C or its analog, 3,4-dehydroproline, produced a greater effect on gene expression than 1 mm l-Pro or 75 mm NaCl. These chemicals did not induce hsp70, S-adenosylmethionine synthetase, or another osmotically induced gene, Em, to any significant extent. Unlike NaCl, gene induction by P5C did not depend on the normal levels of either de novo protein synthesis or respiration, and did not raise abscisic acid levels significantly. P5C- and 3,4-dehydroproline-treated plants consumed less O₂, had reduced NADPH levels, had increased NADH levels, and accumulated many osmolytes associated with osmotically stressed rice. These experiments indicate that osmotically induced increases in the concentrations of one or more intermediates in Pro metabolism could be influencing some of the characteristic responses to osmotic stress.     

Surface Ultrastructural Studies on Penetration and Infection Process of Colletotrichum gloeosporioides on Mulberry Leaf Causing Black Spot Disease

Unicelled conidia of Colletotrichum gloeosporioides germinated 3 h after inoculation producing single germ tubes. The orientation of the germ tubes and their lateral branches as they grew was towards the open stomata and away from closed stomata. By 24 h post-inoculation, the lateral branches had developed specialized infection vesicles either over the stomata or within the stomatal cavities. The infection vesicles produced primary infection hyphae which entered the leaves through stomatal openings. The disease symptoms became apparent by 6 days post-inoculation when clusters of abundant conidiophores emerged by rupturing the leaf epidermal layer, forming acervuli mostly near the base of idioblasts. The pressure exerted by the emerging conidiophores caused stretching of epidermal layer leading to the widening of the acervuli. The conidia are borne on the tips of the erect conidiophores.     

I-conotoxin superfamily revisited.

The I-conotoxin superfamily (I-Ctx) is known to have four disulfide bonds with the cysteine arrangement C-C-CC-CC-C-C, and the members inhibit or modify ion channels of nerve cells. Recently, Olivera and co-workers (FEBS J. 2005; 272: 4178-4188) have suggested that the previously described I-Ctx should now be divided into two different gene superfamilies, namely, I1 and I2, in view of their having two different types of signal peptides and exhibiting distinct functions. We have revisited the 28 entries presently grouped as I-Ctx in UniProt Swiss-Prot knowledgebase, and on the basis of in silico analysis have divided them into I1 and I2 superfamilies. The sequence analysis has provided a framework for in silico annotation enabling us to carry out computer-based functional characterization of the UniProtKB/TrEMBL entry Q59AA4 from Conus miles and to predict it as a member of the I2 superfamily. Furthermore, we have predicted the mature toxin of this entry and have proposed that it may be an inhibitor of voltage-gated potassium channels.