Crystal Structure of the Human Fatty Acid Synthase Enoyl-Acyl Carrier Protein-Reductase Domain Complexed with Triclosan Reveals Allosteric Protein-Protein Interface Inhibition

Human fatty acid synthase (FAS) is a large, multidomain protein that synthesizes long chain fatty acids. Because these fatty acids are primarily provided by diet, FAS is normally expressed at low levels; however, it is highly up-regulated in many cancers. Human enoyl-acyl carrier protein-reductase (hER) is one of the FAS catalytic domains, and its inhibition by drugs like triclosan (TCL) can increase cytotoxicity and decrease drug resistance in cancer cells. We have determined the structure of hER in the presence and absence of TCL. TCL was not bound in the active site, as predicted, but rather at the protein-protein interface (PPI). TCL binding induces a dimer orientation change that causes downstream structural rearrangement in critical active site residues. Kinetics studies indicate that TCL is capable of inhibiting the isolated hER domain with an IC₅₀ of ∼55 μm. Given the hER-TCL structure and the inhibition observed in the hER domain, it seems likely that TCL is observed in the physiologically relevant binding site and that it acts as an allosteric PPI inhibitor. TCL may be a viable scaffold for the development of anti-cancer PPI FAS inhibitors.     

High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization

Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.     

Cold-adapted and rhizosphere-competent strain of Rahnella sp. with broad-spectrum plant growth-promotion potential

A phosphate-solubilizing bacterial strain isolated from Hippophae rhamnoides rhizosphere was identified as Rahnella sp. based on its phenotypic features and 16S rRNA gene sequence. The bacterial strain showed the growth characteristics of a cold-adapted psychrotroph, with the multiple plant growth-promoting traits of inorganic and organic phosphate solubilization, 1-aminocyclopropane-1- carboxylate-deaminase activity, ammonia generation, and siderophore production. The strain also produced indole- 3-acetic acid, indole-3-acetaldehyde, indole-3-acetamide, indole-3-acetonitrile, indole-3-lactic acid, and indole-3- pyruvic acid in tryptophan-supplemented nutrient broth. Gluconic, citric and isocitric acids were the major organic acids detected during tricalcium phosphate solubilization. A rifampicin-resistant mutant of the strain exhibited high rhizosphere competence without disturbance to the resident microbial populations in pea rhizosphere. Seed bacterization with a charcoal-based inoculum significantly increased growth in barley, chickpea, pea, and maize under the controlled environment. Microplot testing of the inoculum at two different locations in pea also showed significant increase in growth and yield. The attributes of coldtolerance, high rhizosphere competence, and broad-spectrum plant growth-promoting activity exhibited the potential of Rahnella sp. BIHB 783 for increasing agriculture productivity.     

Critique on the Use of the Standardized Avian Acute Oral Toxicity Test for First Generation Anticoagulant Rodenticides

Avian risk assessments for rodenticides are often driven by the results of standardized acute oral toxicity tests without regards to a toxicant's mode of action and time course of adverse effects. First generation anticoagulant rodenticides (FGARs) generally require multiple feedings over several days to achieve a threshold concentration in tissue and cause adverse effects. This exposure regimen is much different than that used in the standardized acute oral toxicity test methodology. Median lethal dose values derived from standardized acute oral toxicity tests underestimate the environmental hazard and risk of FGARs. Caution is warranted when FGAR toxicity, physiological effects, and pharmacokinetics derived from standardized acute oral toxicity testing are used for forensic confirmation of the cause of death in avian mortality incidents and when characterizing FGARs’ risks to free-ranging birds.     

Toxoplasma gondii infection reduces predator aversion in rats through epigenetic modulation in the host medial amygdala

Male rats (Rattus novergicus) infected with protozoan Toxoplasma gondii relinquish their innate aversion to the cat odours. This behavioural change is postulated to increase transmission of the parasite to its definitive felid hosts. Here, we show that the Toxoplasma gondii infection institutes an epigenetic change in the DNA methylation of the arginine vasopressin promoter in the medial amygdala of male rats. Infected animals exhibit hypomethylation of arginine vasopressin promoter, leading to greater expression of this nonapeptide. The infection also results in the greater activation of the vasopressinergic neurons after exposure to the cat odour. Furthermore, we show that loss of fear in the infected animals can be rescued by the systemic hypermethylation and recapitulated by directed hypomethylation in the medial amygdala. These results demonstrate an epigenetic proximate mechanism underlying the extended phenotype in the Rattus novergicus–Toxoplasma gondii association.     

Effects of moderate hypothermia on O2 consumption at various O2 deliveries in a sheep model.

The effects of modest hypothermia on oxygen consumption (VO2) were studied at various levels of oxygen delivery (DO2) in six sheep. Each animal was placed on cardiopulmonary bypass by extrathoracic cannulations. DO2 was varied by changing blood flow through an extracorporeal circuit. VO2 was measured spirometrically across a membrane lung. VO2 was initially measured at various levels of DO2 at normothermic temperatures (39 degrees C). The animals were then cooled to 33 degrees C. DO2 was varied, and the corresponding VO2's were determined. The data at both temperatures demonstrated the biphasic relationship of VO2 to various levels of DO2. A critical level of DO2 (DO2 crit) was defined to reflect the transition area between the dependent and independent portions of the consumption-delivery curve. The average baseline VO2's on the delivery independent portion of the curve were calculated to be 5.33 and 3.17 ml O2.kg-1.min-1 at 39 and 33 degrees C, respectively (P less than 0.001). The corresponding DO2 crit's were 6.17 and 4.57 ml O2.kg-1.min-1 (P less than 0.05). The oxygen extraction ratios at DO2 crit for each of these temperatures did not differ significantly. We conclude that hypothermia, by lowering baseline VO2, reduces DO2 crit. Hypothermia may therefore reduce or eliminate the anaerobic metabolism and subsequent acidosis that would otherwise occur during normothermia at low levels of DO2.     

Accelerating public sector rice breeding with high-density KASP markers derived from whole genome sequencing of <Emphasis Type="Italic">indica</Emphasis> rice

Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on >?18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.     

Precise transfers of genes for high grain iron and zinc from wheat-<Emphasis Type="Italic">Aegilops</Emphasis> substitution lines into wheat through pollen irradiation

Nearly 2 billion people worldwide are suffering from iron (Fe) deficiency anemia and zinc (Zn) deficiency. The available elite bread wheat cultivars have inherently low grain micronutrient content. Biofortification for grain Fe and Zn content is one of the most feasible and cost-effective approach for combating widespread deficiency of the micronutrients. QTL controlling high grain Fe and Zn have been mapped on groups 2 and 7 chromosomes of Triticeae. The present study was initiated for precise transfers of genes for high grain Fe and Zn on group 2 and 7 chromosomes of wheat-Aegilops substitution lines to wheat cultivars using pollen radiation hybridization. The pollen radiation hybrids (PRH₁) derived from 1.75 krad irradiated spikes showed the presence of univalents and multivalents in meiotic metaphase-I indicating the effectiveness of radiation dose. In the advanced generation PRH₅, the plants selected with stable chromosome number and high grain Fe and Zn content were analyzed with wheat groups 2 and 7 chromosome specific intron targeted amplified polymorphism (ITAP) markers of the metal homeostasis genes to monitor the transfers of alien genes from the substituted Aegilops chromosomes. The group 2 chromosome derivatives showed the presence of NAS2, FRO2, VIT1, and ZIP2 Aegilops genes whereas the group 7 derivatives had YSL15, NAM, NRAMP5, IRO3, and IRT2 Aegilops genes. The pollen radiation hybrids of both the groups 2 and 7 chromosomes showed more than 30% increase in grain Fe and Zn content with improved yield than the elite wheat cultivar PBW343 LrP indicating small and compensating transfers of metal homeostasis genes of Aegilops into wheat.     

A Ewing's Sarcoma Cell Line Showing Some, but Not All, of the Traits of a Cholinergic Neuron

Abstract: The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 ± 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high-affinity, hemicholinium-3-sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High-affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15-kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium-dependent release but lacks an organized release mechanism.