Hypercholesterolemia Enhances Tolerance to Lethal Systemic Hypoxia in Middle-Aged Mice: Possible Role of VEGF Downregulation in Brain

Hypercholesterolemia (HCL) is commonly associated with impaired vascular relaxation response and augmented vasoconstriction. Interestingly, it was shown that animals with HCL were less vulnerable to seizures and several clinical studies also revealed a better outcome after stroke in the patients with HCL. To this context, the present study was designed to test the hypothesis that HCL would enhance the animals' resistance to severe systemic hypoxia and in turn prolong their survival time under such noxious condition. Four groups of middle-aged (mean age: 51.1 ± 2.8 weeks) male C57BL/6J wild-type mice (C57BL-WT) and low-density lipoprotein receptor knockout mice (LDLR-KO) were included in the study: two groups were exposed to severe normobaric hypoxia (5% F_IO₂) and other two groups were used for brain tissue sample collection and Western blot analysis. The survival time under the hypoxic condition was recorded for each animal. Individual blood samples were collected immedtately after the cessation of spontaneous breathing for measuring plasma total cholesterol (TCL) and triglycerides. The results show that the hypoxia survival time was longer in LDLR-KO than C57BL-WT (i.e. 3.7 ± 0.5 versus 2.3 ± 0.2 min; P < 0.05). A positive correlation was found between TCL and the survival time (r ² = 0.43; P < 0.05). Furthermore, a significant downregulation of vascular endothelial growth factor (VEGF) was observed in the brain tissue of LDLR-KO, as compared with C57BL-WT (n, = 3/group; P < 0.05), whereas expression of heme oxygenase 1 was similar in these two groups. We conclude that HCL enhances resistance to lethal systemic hypoxia (i.e. 61% increase in survival time) in middle-aged mice. This paradoxical protective effect of HCL was associated with a concomitant downregulation of cerebral VEGF expression, which could potentially blunt the hypoxia-triggered and VEGF-mediated pathophysiological events leading to death.     

Structure determination of symmetric homo-oligomers by a complete search of symmetry configuration space, using NMR restraints and van der Waals packing

Structural studies of symmetric homo-oligomers provide mechanistic insights into their roles in essential biological processes, including cell signaling and cellular regulation. This paper presents a novel algorithm for homo-oligomeric structure determination, given the subunit structure, that is both complete, in that it evaluates all possible conformations, and data-driven, in that it evaluates conformations separately for consistency with experimental data and for quality of packing. Completeness ensures that the algorithm does not miss the native conformation, and being data-driven enables it to assess the structural precision possible from data alone. Our algorithm performs a branch-and-bound search in the symmetry configuration space, the space of symmetry axis parameters (positions and orientations) defining all possible C(n) homo-oligomeric complexes for a given subunit structure. It eliminates those symmetry axes inconsistent with intersubunit nuclear Overhauser effect (NOE) distance restraints and then identifies conformations representing any consistent, well-packed structure to within a user-defined similarity level. For the human phospholamban pentamer in dodecylphosphocholine micelles, using the structure of one subunit determined from a subset of the experimental NMR data, our algorithm identifies a diverse set of complex structures consistent with the nine intersubunit NOE restraints. The distribution of determined structures provides an objective characterization of structural uncertainty: backbone RMSD to the previously determined structure ranges from 1.07 to 8.85 A, and variance in backbone atomic coordinates is an average of 12.32 A(2). Incorporating vdW packing reduces structural diversity to a maximum backbone RMSD of 6.24 A and an average backbone variance of 6.80 A(2). By comparing data consistency and packing quality under different assumptions of oligomeric number, our algorithm identifies the pentamer as the most likely oligomeric state of phospholamban, demonstrating that it is possible to determine the oligomeric number directly from NMR data. Additional tests on a number of homo-oligomers, from dimer to heptamer, similarly demonstrate the power of our method to provide unbiased determination and evaluation of homo-oligomeric complex structures.     

Introduction of bacterial blight resistance into Triguna,a high yielding,mid-early duration rice variety

Bacterial blight (BB) is a serious disease of rice in India. We have used molecular marker-assisted selection in a backcross breeding program to introgress three genes (Xa21, xa13, and xa5) for BB resistance into Triguna, a mid-early duration, high yielding rice variety that is susceptible to BB. At each generation in the backcross program, molecular markers were used to select plants possessing these resistance genes and to select plants that have maximum contribution from the Triguna genome. A selected BC3F1 plant was selfed to generate homozygous BC₃F₂ plants with different combinations of BB resistance genes. Plants containing the two-gene combination, Xa21 and xa13, were found to exhibit excellent resistance against BB. Single plant selections for superior agronomic characteristics were performed on the progeny of these plants, from BC₃F₃ generation onwards. The selected plants were subjected to yield trials at the BC₃F₈ generation and were found to have a significant yield advantage over Triguna. The newly developed lines are being entered into national multi-location field trials. This work represents a successful example of the application of molecular marker-assisted selection for BB resistance breeding in rice.     

Development of a simple functional marker for fragrance in rice and its validation in Indian Basmati and non-Basmati fragrant rice varieties

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.     

Anti-neuroinflammatory activity of 1,5-benzodiazepine derivatives

A series of 2,3-dihydro-1,5-benzodiazepines were synthesized and evaluated for anti-inflammatory effects in microglia cells. Among the 1,5-benzodiazepines tested, compound 3e strongly inhibited LPS-induced nitric oxide (NO) production, with an IC₅₀ value of 7.0 μM in the microglia cells. Also, compound 3e significantly inhibited the enzymatic activity of inducible NO synthase (iNOS) without changes in iNOS protein expression or NO scavenging activity. This result suggests that compound 3e showed anti-neuroinflammatory effects by suppressing iNOS enzyme activity.     

Development and validation of a PCR-based functional marker system for the major wide-compatible gene locus S5 in rice

The major wide-compatibility gene locus S5 in rice (Oryza sativa L.) located on chromosome 6 has been recently cloned and a 136-bp deletion in the candidate gene encoding aspartyl protease has been characterized to be specific for wide-compatible varieties, while many single nucleotide polymorphisms have been identified at S5 between indica and japonica rice types. In the present study, we designed a PCR-based multiplex functional marker system targeting the deletion and the SNPs for precise determination of the allelic status at S5. By deploying the marker system, the allelic status at the S5 locus in 584 rice genotypes has been assayed. A total of 116 genotypes, including 11 cultivars, two known wide-compatible varieties, 48 IRRI germplasm lines, 12 Indian aromatic rice genotypes, 37 restorer lines and six breeding lines, have been identified to possess the 136-bp deletion specific for the neutral allele at S5. The marker system was able to clearly distinguish indica and japonica alleles from the neutral allele and has been validated in a mapping population derived from the three-way cross IR36/Dular//Akihikari, which segregated for spikelet sterility/fertility. The functional marker system targeting S5 developed in the present study will be very useful in rapid identification of wide-compatible genotypes, in predicting the success of inter-subspecific crosses and in targeted introgression of the wide-compatible allele of S5 into elite indica and japonica rice varieties.     

Choline transport for phospholipid synthesis

Choline is an essential nutrient for all cells because it plays a role in the synthesis of the membrane phospholipid components of the cell membranes, as a methyl-group donor in methionine metabolism as well as in the synthesis of the neurotransmitter acetylcholine. Choline deficiency affects the expression of genes involved in cell proliferation, differentiation, and apoptosis, and it has been associated with liver dysfunction and cancer. Abnormal choline transport and metabolism have been implicated in a number of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Therefore, the study of choline transport and the characteristics of choline transporters are of central importance to understanding the mechanisms that underlie membrane integrity and cell signaling in such disorders. Kinetic studies with radiolabeled choline and inhibitors distinguish three systems for choline transport: (i) low-affinity facilitated diffusion, (ii) high-affinity, Na+-dependent transport, and (iii) intermediate-affinity, Na+-independent transport. It is only recently, however, that the proteins having transport characteristics of at least one of these systems have been identified. They include (i) polyspecific organic cation transporters (OCTs) with low affinity for choline, (ii) high-affinity choline transporters (CHT1s), and (iii) intermediate-affinity choline transporter-like (CTL1) proteins. CHT1 and CTL1 but not OCT transporters are selectively inhibited with hemicholinium-3 and essentially display characteristics of specialized transporters for targeted choline metabolism. CHT1 is abundant in neurons and almost exclusively supplies choline for acetyl-choline synthesis. The focus here is more on newly-discovered CTL1 choline transporters. They are expressed in different organisms and cell types, apparently not for the biosynthesis of acetylcholine but for the production of the most abundant metabolite of choline, the membrane lipid phosphatidylcholine.     

Geographic Risk for Lyme Disease and Human Granulocytic Ehrlichiosis in Southern New York State

Ixodes scapularis, the tick vector of Lyme disease and human granulocytic ehrlichiosis (HGE), is prevalent in much of southern New York state. The distribution of this species has increased, as have reported cases of both Lyme disease and HGE. The unreliability of case reports, however, demonstrates the need for tick and pathogen surveillance in order to accurately define areas of high risk. In this study, a total of 89,550 m² at 34 study sites was drag sampled in 1995 and a total of 51,540 m² at 40 sites was sampled in 1996 to determine tick and pathogen distribution in southern New York state. I. scapularis was collected from 90% of the sites sampled, and regionally, a 2.5-fold increase in nymphal abundance occurred from 1995 to 1996. I. scapularis individuals from all sites were infected with Borrelia burgdorferi in 1995, while an examination of ticks for both B. burgdorferi and the agent of HGE in 1996 confirmed that these organisms were present in all counties; the average coinfection rate was 1.9%. No correlation was found between estimated risk and reported cases of Lyme disease. The geographic disparity of risk observed among sites in this study underscores the need for vector and pathogen surveillance on a regional level. An entomologic risk index can help identify sites for targeted tick control efforts.     

Ethanol modulates the VR-1 variant amiloride-insensitive salt taste receptor. II. Effect on chorda tympani salt responses

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by direct measurement of intracellular Na(+) activity ([Na(+)](i)) using fluorescence imaging in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. CT responses to KCl and NaCl were recorded in Sprague-Dawley rats, and in wild-type (WT) and vanilloid receptor-1 (VR-1) knockout mice (KO). CT responses were monitored in the presence of Bz, a specific blocker of the epithelial Na(+) channel (ENaC). CT responses were also recorded in the presence of agonists (resiniferatoxin and elevated temperature) and antagonists (capsazepine and SB-366791) of VR-1 that similarly modulate the Bz-insensitive VR-1 variant salt taste receptor. In the absence of mineral salts, ethanol induced a transient decrease in TRC volume and elicited only transient phasic CT responses. In the presence of mineral salts, ethanol increased the apical cation flux in TRCs without a change in volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a phasic component and a sustained tonic component. At concentrations <50%, ethanol enhanced responses to KCl and NaCl, while at ethanol concentrations >50%, those CT responses were inhibited. Resiniferatoxin and elevated temperature increased the sensitivity of the CT response to ethanol in salt-containing media, and SB-366791 inhibited the effect of ethanol, resiniferatoxin, and elevated temperature on the CT responses to mineral salts. VR-1 KO mice demonstrated no Bz-insensitive CT response to NaCl and no sensitivity to ethanol. We conclude that ethanol increases salt taste sensitivity by its direct action on the Bz-insensitive VR-1 variant salt taste receptor.