Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium

The objective was to evaluate the parthenogenetic activation of domestic cat oocytes. Cumulus-oocyte complexes matured for 36 h were subjected to three protocols of parthenogenetic activation: Group 1 - ionomycin + cycloheximide; Group 2 - ionomycin + roscovitine; and Group 3 - ionomycin + strontium. As a control, a fourth group of oocytes were cultured in the absence of any activation agent. In all groups, embryos were cultured in SOFaa for 72 h after activation and evaluated for activation rate, cleavage, and embryonic development using Hoechst33342. There were no significant differences among the three treated groups for rates of activated oocytes (70.1 ± 4.3, 75.5 ± 4.7, and 61.9 ± 7.2%, for Treatments 1, 2, and 3 respectively; mean ± SEM), or cleavage (48.1 ± 5.9, 47.4 ± 3.8, and 33.3 ± 6.8%). However, activation and cleavage rates were higher (P < 0.05) than those in the control group (35.5 ± 6.4 and 11.8 ± 4.0%). There were no significant differences among treatment groups for proportion of embryos with 2-10 cells, 10-16 cells, and morulas. In the Control group, the embryo production rate was lower (P < 0.05), although the activation rate was high. The authors concluded that all three treatments effectively induced parthenogenetic activation of domestic cat oocytes. However, to optimize the use of strontium and roscovitine, a dose response and the effect of the presence of Ca⁺⁺ in the medium requires further study.     

Solid-state NMR studies of a diverged microsomal amino-proximate delta12 desaturase peptide reveal causes of stability in bilayer: tyrosine anchoring and arginine snorkeling

This study reports the solid-state NMR spectroscopic characterization of the amino-proximate transmembrane domain (TM-A) of a diverged microsomal delta12-desaturase (CREP-1) in a phospholipid bilayer. A series of TM-A peptides were synthesized with 2H-labeled side chains (Ala-53, -56, and -63, Leu-62, Val-50), and their dynamic properties were studied in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) bilayers at various temperatures. At 6 mol % peptide to lipid, 31P NMR spectra indicated that the peptides did not significantly disrupt the phospholipid bilayer in the L(alpha) phase. The 2H NMR spectra from Ala-53 and Ala-56 samples revealed broad Pake patterns with quadrupolar splittings of 16.9 kHz and 13.3 kHz, respectively, indicating restricted motion confined within the hydrocarbon core of the phospholipid bilayer. Conversely, the deuterated Ala-63 sample revealed a peak centered at 0 kHz with a linewidth of 1.9 kHz, indicating increased side-chain motion and solvent exposure relative to the spectra of the other Ala residues. Val-50 and Leu-62 showed Pake patterns, with quadrupolar splittings of 3.5 kHz and 3.7 kHz, respectively, intermediate to Ala-53/Ala-56 and Ala-63. This indicates partial motional averaging and supports a model with the Val and Leu residues embedded inside the lipid bilayer. Solid-state NMR spectroscopy performed on the 2H-labeled Ala-56 TM-A peptide incorporated into magnetically aligned phospholipid bilayers indicated that the peptide is tilted 8 degrees with respect to the membrane normal of the lipid bilayer. Snorkeling and anchoring interactions of Arg-44 and Tyr-60, respectively, with the polar region or polar hydrophobic interface of the lipid bilayer are suggested as control elements for insertional depth and orientation of the helix in the lipid matrix. Thus, this study defines the location of key residues in TM-A with respect to the lipid bilayer, describes the conformation of TM-A in a biomembrane mimic, presents a peptide-bilayer model useful in the consideration of local protein folding in the microsomal desaturases, and presents a model of arginine and tyrosine control of transmembrane protein stability and insertion.     

Atypical Biosynthetic Properties of a Δ12/ν+3 Desaturase from the Model Basidiomycete Phanerochaete chrysosporium

The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Δ¹²-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C₁₈ substrates with ν+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (ν). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30°C versus 15°C). Thus, while maintaining the hallmark activity of the fatty acyl Δ¹²-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.Desaturases, the enzymes responsible for unsaturated fatty acid biosynthesis, are found throughout the eukaryotic taxa. Critical cellular processes dependent on the modification of acyl lipids by desaturases include the regulation of membrane structure and fluidity, proper function of ion channels and other membrane proteins, and the biosynthesis of signaling molecules, such as jasmonic acid and arachidonic acid-derived second messengers (53, 71). Polyunsaturated fatty acids (PUFAs) with double bonds at carbon-12, such as linoleic acid (18:2Δ9c,12c), are not synthesized by animals, who therefore depend upon the activities of the stepwise action of the Δ⁹- and Δ¹²-desaturases from plants and lower eukaryotes to generate these essential lipids.Supplementation of our diet with PUFAs derived from transgenic organisms has been targeted in recent years. Expression of fungal (37) and plant (56) desaturase genes in mammalian cells has been explored as a means to enhance the nutritional quality of meat products. Oleate and PUFA desaturases and elongases are gene targets sought after for transgenic production of the C₂₀ and C₂₂ polyunsaturated food supplements docosahexenoic and eicosapentenoic acids in alga, plants, and yeast (35, 51). The practical success of lipid metabolic engineering studies is dependent upon the expression of enzymes with high chemo- and regioselectivity within the transgenic organism, coupled with the manipulation of lipid biochemical flux to result in high, economically viable levels of unsaturated storage oil accumulation.Two evolutionarily distinct desaturase types exist: the soluble plastidal and the membrane-bound endoplasmic reticulum (ER)-localized enzymes, both of which use NAD(P)H and O₂ to sequentially abstract two hydrogens from vicinal sp³-hybridized carbons leading to a cis-alkene (15, 61). Current models for all fatty acyl desaturases postulate the activation of molecular oxygen at a nonheme diferrous active site that culminates with two C-H bond scissions and the formation of water (20, 29, 39, 46). In the case of the microsomal desaturases, conserved iron ligands appear to be located in three distinctive histidine box motifs (61). Microsomal Δ¹²-desaturases (FAD2s) are best known from plants, where they exhibit substantial (60 to 90%) sequence identity and have focused regioselectivity yet have evolved into the diverged desaturases that catalyze distinct oxidative processes, resulting in natural products with hydroxy, conjugated polyalkenyl, epoxy, and acetylenic functionalities (65). Studies of the FAD2 superfamily have been propelled by commercial interest in the modification of standard oilseed crops with “diverged” fad2 genes that show atypical regio- and/or chemoselectivity (32, 38, 50). Despite the sheer number of plant Δ¹²-desaturases, the refractory nature of these membrane-bound enzymes to purification has left structure/function relationships ill defined (33). Consequently, only the rough classification of the FAD2 enzymes into distinct functional or evolutionary classes has occurred, largely using genetic and in vivo functional characterization (12, 38, 42).While the Δ¹²- or FAD2 desaturases, which form a 12,13-double bond, are best known from plants, fungal fad2 homologs have been found in zygomycetes and ascomycetes (8, 52, 59). As suggested by molecular clock data, Basidiomycota and Ascomycotina diverged approximately 550 million years ago (3), indicating that metabolic basidiomycete genes may differ significantly from those of other fungal subtypes. With their high linoleic acid content, typically 60 to 80% of the lipid in basidiomycete fruiting bodies (63), and their ability to grow under varied temperature regimes, macrofungi provide an untapped genetic resource for desaturases that may be well suited for biotechnological applications. Indeed, two homobasidiomycete Δ¹²-desaturases have been recently reported (57, 77).In fungi, variations in membrane lipid composition caused by temperature cycling may be integral to the morphological changes of fruit body formation (58). Linoleate-derived hydroxy fatty acids and lactones have been shown to provide molecular signals, called Psi factors, involved in ascomycete sporulation (8, 9). Disruption of the oleoyl-phosphatidycholine desaturase odeA in Aspergillus parasiticus results in diminished growth; delayed germination has been proposed as a countermeasure for controlling this aflatoxigenic species (74). Additionally, volatile organic species emitted by fungi (e.g., (−)-1-octen-3-ol and 10-oxodecanoic acid) play a role in the palatability of mushrooms and may also mediate sporulation and the transition from vegetative to reproductive tissues (10). Separately, targeting Δ¹²-desaturases, which have no known homologs in humans, in pathogenic basidiomycetes has real potential as selective fungicidal targets. Cryptococcus neoformans infections in AIDS and immunosuppressed patients are frequently observed in the clinic; consequently, developing antimicrobial agents targeting C. neoformans will markedly improve the health of these patients (55).Phanerochaete chrysosporium is a widely distributed wood decay homobasidiomycete that has become a model system for studying lignocellulose degradation (41). It harbors an array of peroxidases and degrading lignocellulose as well as aromatic pollutants (14, 26). A role for linoleate (18:2), which may be supplied from endogenous wood lipids or through fungal Δ¹²-desaturation, in the mediation of lignin degradation has been suggested whereby diffusible lipid-derived peroxyl or alkoxy radicals aid in the initial decay of sound wood, particularly in white-rot fungi lacking lignin peroxidase (36, 73). The production of free 18:2 during early colonization of wood meal, followed by extracellular lipid peroxidation and in vitro degradation of nonphenolic lignin, has been shown for the white-rot fungus Ceriporiopsis subvermispora (16).As part of our program to elucidate the biosynthetic networks leading to highly unsaturated natural products in basidiomycetes (e.g., the polyacetylenes) (45), we carried out the cloning and sequence analysis of the gene encoding the sole Δ¹²-desaturase from P. chrysosporium. In this paper, we demonstrate its function through heterologous expression in Saccharomyces cerevisiae and show that this enzyme has features distinct from other fungal and plant FAD2 desaturases, which should facilitate future isolation and structure-function analysis of diverged macrofungal desaturases.     

Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising ∼70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria.Plague is a disease of historical epidemics that remains an important public health problem in limited areas of the world (1). Disease transmission usually occurs through transfer of the bacillus Yersinia pestis by the bite of a flea. However, less frequent direct transfer of viable bacteria by respiratory droplets may result in primary pneumonic infection. A transient intracellular infection of phagocytic cells (2) occurs during the earliest stage of bubonic plague followed by rapid extracellular expansion of bacteria in lymph nodes. The prototypical lymphatic infection of bubonic plague may also progress to bacteremic or pneumonic infection with a very high rate of fatality if there is not rapid intervention by antibiotic treatment (3). Among the reported cases occurring annually in the United States, 15% were fatal in 2006 (4). Although only small numbers of human cases occur each year in North America, a more substantial incidence of plague is found in wild animal populations (5) with seroprevalence rates of up to 100% among mammalian carnivores in endemic areas (6). The geographic range of infection within feral populations is presently unknown but may contribute significantly to the reservoir of potential disease transmission to humans.Diagnostic tests and prophylactic vaccines or therapies must rapidly distinguish or protect against the many infectious diseases that present similar initial symptoms. Specific diagnostic tests and vaccines for plague are public health priorities primarily because of the threat from potential acts of terrorism. Because human deaths may occur within 48 h of infection (7), delays in proper diagnosis have led to disease complications and fatalities from plague (8). Yet the identification of bacterial sepsis at the earliest stage of clinical presentation is challenging because of the generalized nature of disease symptoms and the difficulty in culturing infectious agents or isolating sufficient material to identify the infectious agent by amplification of genetic markers. Although host antibody responses provide a sensitive indicator of current or past infection, insufficient numbers of validated biomarkers are available, and extensive antibody cross-reactivity among Gram-negative pathogens (9–12) complicates the direct analysis of serum.Identification of plague-specific antibody interactions is a daunting task because of the complexity of the bacterial proteome encountered by the host during infection. The chromosome of Y. pestis CO92 encodes ∼3885 proteins, whereas an additional 181 are episomally expressed by pCD1, pMT1, and pPCP1. For comparison, the proteome of Y. pestis KIM¹ contains 4202 individual proteins (13), 87% in common with CO92 (14), and the closely related enteric pathogen Yersinia pseudotuberculosis (15, 16) contains ∼4038 proteins (chromosome plus plasmids). Recent technical advances have facilitated the development of microarrays comprising full-length, functional proteins that represent nearly complete proteomes. For example, Zhu et al. (17) reported the development of a proteome microarray containing the full-length, purified expression products of over 93% of the 6280 protein-coding genes of the yeast Saccharomyces cerevisiae, and Schmid et al. (18) described the human antibody repertoire for vaccinia virus recognition by using a viral proteome microarray. This approach opens the possibility of examining the entire bacterial proteome to elucidate proteins or protein pathways that are essential to pathogenicity or host immunity. We sought to identify biomarkers that could distinguish plague from diseases caused by other bacterial pathogens by measuring host antibody recognition of individual proteins contained within the Y. pestis proteome. The previously reported genomic sequences of Y. pestis strains KIM (13) and CO92 (14), sharing 95% identity, were used for reference. Approximately 77% of the putative Y. pestis proteome can be classified by known homologies. We successfully expressed and purified the majority (70%) of the 4066 ORFs encoded by the chromosome and plasmids of Y. pestis KIM and arrayed these products onto glass slides coated with nitrocellulose. The Y. pestis ORFs subcloned into expression vectors were fully sequenced to confirm quality and identity before use. Different approaches for studying the antibody repertoire for plague in rabbits and non-human primates were compared. Based on results from experiments using the Y. pestis proteome microarray, we identified new candidates for antibody biomarkers of bacterial infections and patterns of cross-reactivity that may be useful diagnostic tools.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass

In this study an industrial Saccharomyces cerevisiae yeast strain capable of fermenting ethanol from pretreated lignocellulosic material was engineered. Genes encoding cellulases (endoglucanase, exoglucanase and β-glucosidase) were integrated into the chromosomal ribosomal DNA and delta regions of a derivative of the K1-V1116 wine yeast strain. The engineered cellulolytic yeast produces ethanol in one step through simultaneous saccharification and fermentation of pretreated biomass without the addition of exogenously produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fermented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v. These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process.     

Predator decline leads to decreased stability in a coastal fish community

Fisheries exploitation has caused widespread declines in marine predators. Theory predicts that predator depletion will destabilise lower trophic levels, making natural communities more vulnerable to environmental perturbations. However, empirical evidence has been limited. Using a community matrix model, we empirically assessed trends in the stability of a multispecies coastal fish community over the course of predator depletion. Three indices of community stability (resistance, resilience and reactivity) revealed significantly decreasing stability concurrent with declining predator abundance. The trophically downgraded community exhibited weaker top‐down control, leading to predator‐release processes in lower trophic levels and increased susceptibility to perturbation. At the community level, our results suggest that high predator abundance acts as a stabilising force to the naturally stochastic and highly autocorrelated dynamics in low trophic species. These findings have important implications for the conservation and management of predators in marine ecosystems and provide empirical support for the theory of predatory control.     

The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase

The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of drugs used both in the treatment of acute leukaemia (6-mercaptopurine and 6-thioguanine) and as an immunosuppressant in patients with autoimmune diseases or following organ transplantation (azathioprine). Studies of enzyme activity in red blood cells have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. Patients with biallelic mutations and undetectable enzyme activity suffer life-threatening myelosuppression when treated with conventional doses of these drugs. Patients with intermediate activity have an increased risk of drug-associated toxicity. In the Caucasian populations studied to date, intermediate activity is associated with mutations at two sites of the TPMT gene, G460A and A719G (designated TPMT*3A), in 80% of cases. Detection of these mutations has, to date, been based on the analysis of restriction digests of PCR products. In order to simplify this process we have investigated the ability of denaturing high pressure liquid chromatography (DHPLC) to detect the A719G mutation. DHPLC of PCR products from 15 known heterozygotes (TPMT*3A/TPMT*1) and 18 known homozygotes (TPMT*1/TPMT*1) gave a clear pattern difference between the groups and 100% concordance with the results of restriction digests. These results suggest DHPLC represents a valuable technique for accurate and rapid detection of pharmacologically important mutations in the TPMT gene.