Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.     

Investigation of a His-rich arabinogalactan-protein for micronutrient biofortification of cereal grain

The micronutrient content of most cereal grains is low and responsible for malnutrition deficiencies in millions of people who rely on grains as their primary food source. Any strategy that can increase the micronutrient content of grain will have significant benefits to world health. We identified a gene from barley encoding a cell wall protein with multiple histidine (His)-rich motifs interspersed with short arabinogalactan-protein (AGP) domains and have called it Hordeum vulgare His-rich AGP (HvHRA1). Sequence analysis shows that His-rich AGPs are rare in plants and that the number of His-rich and AGP domains differ between cereals and dicots. The barley and wheat encoded proteins have more than 13 His-rich domains, whereas the putative rice orthologue has only 5 His-rich regions. His-rich motifs are well-established metal-binding motifs; therefore, we developed transgenic (Tx) rice plants that constitutively overexpress barley HvHRA1. There was no significant effect on plant growth or grain yield in Tx plants. Purification of AGPs from wild-type and Tx plants showed that only Tx plants contained detectable levels of a His-rich AGP. Calcein assay shows that the AGP fraction from Tx plants had increased binding affinity for Cu(2+) . Micronutrient analysis of brown and white rice showed that the grain nutrient yield for Fe, Zn and Cu was higher in two Tx lines compared to their respective nulls, although the differences were not statistically significant. This approach highlights the potential of the plant apoplast (cell wall) for storage of key nutrients through overexpression of genes for metal-binding proteins.     

The control of boron accumulation by two genotypes of wheat

The mechanism of boron (B) uptake in wheat was studied using two genotypes with known differences in their ability to accumulate B. Influx and efflux of B was measured in the roots of intact 21 d old plants.Roots grown in 15 M B, when transferred to solutions containing 1mM B showed a rapid increase in B content for up to 60 min, after which no further increase was evident up to 4 h. No genotypic difference in B influx was apparent over these time periods. Roots grown in 1mM B for 7 d and then rinsed in B-free solutions quickly lost most of B that they contained within 1 hour; little further efflux was observed over the following three hours. As with the influx, no genotypic difference in B flux was evident.It is suggested that the lack of genotypic difference in the short-term B fluxes could be due to a masking effect of extracellular B bound in the cell walls of the roots.Department of Botany, University of Adelaide     

HKT1;5-like cation transporters linked to Na+ exclusion loci in wheat, Nax2 and Kna1

Bread wheat (Triticum aestivum) has a greater ability to exclude Na⁺ from its leaves and is more salt tolerant than durum wheat (Triticum turgidum L. subsp. durum [Desf.]). A novel durum wheat, Line 149, was found to contain a major gene for Na⁺ exclusion, Nax2, which removes Na⁺ from the xylem in the roots and leads to a high K⁺-to-Na⁺ ratio in the leaves. Nax2 was mapped to the distal region on chromosome 5AL based on linkage to microsatellite markers. The Nax2 locus on 5AL coincides with the locus for a putative Na⁺ transporter, HKT1;5 (HKT8). The Nax2 region on 5AL is homoeologous to the region on chromosome 4DL containing the major Na⁺ exclusion locus in bread wheat, Kna1. A gene member of the HKT1;5 family colocates to the deletion bin containing Kna1 on chromosome 4DL. This work provides evidence that Nax2 and Kna1 are strongly associated with HKT1;5 genes.     

Multilocus sequence types of Finnish bovine <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> isolates and their attribution to human infections

Background  

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.     

吐鲁番市胃食管反流病发生相关危险因素分析（附280例报道）

目的：探讨280例胃食管反流病（GERD）的分布特点及危险因素。方法：对临床诊断和胃镜确诊的280例GERD患者进行临床和风险因子相关性分析。结果：不论汉族还是维族,男性患者比例均明显高于女性;汉族患者高发年龄段早于维族患者（z=-2．939,P=0．003,）;汉族和维族患者占反流性食管炎和Barrett食管比例分别为42．4％、81_3％及56．5％、18．8％,其中汉族患者Barrett食管比例较高（X2=14．358,P=0．000）;肥胖、习惯性便秘、重体力活动者、饮食习惯不良在维族患者中的比例较高（P〈0．001）。结论：GERD与性别、年龄密切相关,男性多于女性,汉族患者发病年龄高峰旱于维族患者;汉族患者Barrett食管发生比例高于维族患者;肥胖、习惯性便秘、重体力活动、饮食习惯不良可能是GERD尤其是维族人群GERD的危险因素。     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm

Background

Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g⁻¹ Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g⁻¹ Fe in endosperm.

Methodology/Principal Findings

Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g⁻¹ Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g⁻¹ Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.

Conclusions

The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.