Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (−8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (−8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.     

Detection of ricin using a carbon nanofiber based biosensor

We report ricin detection using antibody and aptamer probes immobilized on a nanoelectrode array (NEA) consisting of vertically aligned carbon nanofibers (VACNFs). These biosensor chips are fabricated on a wafer scale using steps common in integrated circuit manufacturing. Electrochemical impedance spectroscopy is used to characterize the detection event and the results indicate that the electron transfer resistance changes significantly after the ricin protein binds to the probe. Further confirmation is obtained from evaluation of the electrode surface by atomic force microscopy which clearly shows a change in height from the bare electrode to the surface bound by the probe-protein.     

Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.     

Extra-Lysosomal Localization of Arylsulfatase B in Human Colonic Epithelium

The enzyme arylsulfatase B (N-acetylgalactosamine-4-sulfatase; ARSB; ASB) removes 4-sulfate groups from the sulfated glycosaminoglycans (sGAG) chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Inborn deficiency of ARSB leads to the lysosomal storage disease mucopolysaccharidosis VI, characterized by accumulation of sGAG in vital organs, disruption of normal physiological processes, severe morbidity, and premature death. Recent published work demonstrated extra-lysosomal localization with nuclear and cell membrane ARSB observed in bronchial and colonic epithelial cells, cerebrovascular cells, and hepatic cells. In this report, the authors present ARSB immunostaining in a colonic microarray and show differences in distribution, intensity, and pattern of ARSB staining among normal colon, adenomas, and adenocarcinomas. Distinctive, intense luminal membrane staining was present in the normal epithelial cells but reduced in the malignancies and less in the grade 3 than in the grade 1 adenocarcinomas. In the normal cores, a distinctive pattern of intense cytoplasmic positivity at the luminal surface was followed by reduced staining deeper in the crypts. ARSB enzymatic activity was significantly greater in normal than in malignant tissue. These study findings affirm extra-lysosomal localization of ARSB and suggest that altered ARSB immunostaining and reduced activity may be useful indicators of malignant transformation in human colonic tissue.     

Analysis of transgene(s) (psy+crtI) inheritance and its stability over generations in the genetic background of indica rice cultivar Swarna

Rice the major staple food crop which feeds more than half of the world’s population but, lacks pathway to synthesize and accumulate provitamin A in endosperm therefore rice eaters particularly children, and pregnant women suffer due to vitamin A deficiency. The pathway for provitamin A synthesis in rice endosperm has been engineered and transgenic rice lines have yellow endosperm, called ‘Golden Rice’. The present study aimed at studying the inheritance of transgene(s) in six transgenic events of ‘Golden Rice’ and transfer of provitamin A trait from transgenic lines to a widely grown mega rice variety Swarna. The events E1, R1 and W1 showed normal Mendelian inheritance in F₂, BC₁F₁ and BC₁F₂ generations. The event W1 was studied in BC₁F₃ as well and showed normal Mendelian inheritance of 3:1. The inheritance pattern in L1 event in BC₁F₁ and BC₁F₂ showed normal Mendelian inheritance following expected ratio 1:1 and 3:1 respectively. The two events G1 and T1 showed distorted segregation in BC₁F₂ and BC₂F₂ respectively in Swarna genetic background. In G1 event, transgene inheritance showed segregation distortion in BC₁F₂ in favour of transgene negative plants. In T1 event, inheritance followed expected Mendelian segregation in BC₁F₁, BC₂F₁ and BC₂F₂, generations. However, when tested against co-dominant inheritance 1:2:1 pattern in BC₂F₂, segregation distortion was observed with less than the expected transgene homozygotes. While against 3:1 ratio, it showed the expected segregation pattern in BC₂F₂ generation. Segregation distortion probably due to differential transmission of transgene positive/negative gametes through either/both parents which needs further study.     

Mechanical unbinding of abeta peptides from amyloid fibrils

Using the experimental structures of Abeta amyloid fibrils and all-atom molecular dynamics, we study the force-induced unbinding of Abeta peptides from the fibril. We show that the mechanical dissociation of Abeta peptides is highly anisotropic and proceeds via different pathways when force is applied in parallel or perpendicular direction with respect to the fibril axis. The threshold forces associated with lateral unbinding of Abeta peptides exceed those observed during the mechanical dissociation along the fibril axis. In addition, Abeta fibrils are found to be brittle in the lateral direction of unbinding and soft along the fibril axis. Lateral mechanical unbinding and the unbinding along the fibril axis load different types of fibril interactions. Lateral unbinding is primarily determined by the cooperative rupture of fibril backbone hydrogen bonds. The unbinding along the fibril axis largely depends on the interpeptide Lys-Asp electrostatic contacts and the hydrophobic interactions formed by the Abeta C terminal. Due to universality of the amyloid beta structure, the anisotropic mechanical dissociation observed for Abeta fibrils is likely to be applicable to other amyloid assemblies. The estimates of equilibrium forces required to dissociate Abeta peptide from the amyloid fibril suggest that these supramolecular structures are mechanically stronger than most protein domains.     

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 ± 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa , version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.     

Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli

Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [125I]-iodoazidococaine. Ligand binding using [3H]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol.