Chemical shift based editing of CH<Subscript>3</Subscript> groups in fractionally <Superscript>13</Superscript>C-labelled proteins using GFT (3, 2)D CT-H<Emphasis Type="Underline">CC</Emphasis>H-COSY: stereospecific assignments of CH<Subscript>3</Subscript> groups of Val and Leu residues

We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) (13)C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) (13)C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.     

Phylogeny of the Rodent Genus Isothrix (Hystricognathi, Echimyidae) and its Diversification in Amazonia and the Eastern Andes

Efforts to clarify the affinities of the torós or brush-tailed rats (Isothrix) and document the radiation of these distinctive echimyids have been limited. The discovery of a new Andean species prompted a reanalysis of Isothrix and its relatives. Prior morphological analyses of skulls, mandibles, teeth, and external characters permitted robust diagnosis but offered little resolution of within- or between-group relationships. Analyses of mitochondrial cytochrome b sequences (798 bp), which are available for numerous echimyids, confirm the monophyly of recognized genera, including Isothrix, and resolve a number of interspecific relationships. Strikingly, the Andean toró (Isothrix barbarabrownae) is consistently recovered as sister to the remaining species. These are allied into three clades: I. sinnamariensis + I. pagurus in the lower Amazon Basin and Guianan Shield, I. orinoci + I. negrensis in the Rio Negro and Río Orinoco drainages, and I. bistriata across much of the western and southern Amazon Basin. However, the addition of a new basal taxon does not aid in identifying the sister taxon of Isothrix. These relationships are confirmed in combined analyses of cyt-b with sequence variation in the mitochondrial control region (D-loop; 450 bp) and in the nuclear RAG1 gene (1,072 bp). Analyses identify the Andes, or proto-Andes, as an important theater for the group’s evolution and may offer an explanation for the luxuriant fur of this genus. However, neither the biogeographic history of Isothrix nor the remarkable pelage evolution of the Echimyidae can be understood until the deeper nodes within the arboreal spiny rats (Echimyinae) are more fully resolved.     

Type 1 ribosome-inactivating proteins from <Emphasis Type="Italic">Phytolacca dioica</Emphasis> L. leaves: differential seasonal and age expression,and cellular localization

The expression of type 1 ribosome-inactivating proteins (RIPs) in Phytolacca dioica L. leaves was investigated. Fully expanded leaves of young P. dioica plants (up to 3 years old) expressed two novel RIPs, dioicin 1 and dioicin 2. The former was also found in developing leaves from adult P. dioica within about two and a half weeks after leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint. Fully expanded leaves from adult P. dioica expressed four RIPs (PD-Ls1–4) exhibiting seasonal variation. RIPs were localized in the extracellular space, in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 showed rRNA N-β-glycosidase activity and displayed the following properties, respectively: (1) Mr values of 30,047.00 and 29,910.00, (2) pIs of 8.74 and 9.37, and (3) IC₅₀ values of 19.74 (0.658 nM) and 6.85 ng/mL (0.229 nM). Furthermore, they showed adenine polynucleotide glycosylase activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 had 266 amino acid residues, and the highest percentage identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana, while its identity with other RIPs from Phytolaccaceae was around 40%. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B

Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigations of aspects such as host specificity and transmission patterns require the direct genetic characterisation of parasites from faecal samples. We performed a sequence analysis of four genes (ssrRNA, β-giardin, glutamate dehydrogenase and triose phosphate isomerase) of 61 human isolates and 29 animal isolates. The results showed that multilocus genotypes (MLGs) can be readily defined for G. duodenalis isolates of assemblage A but not for assemblage B. Indeed, for assemblage A isolates, there was no evidence of intra-isolate sequence heterogeneity, and congruent genotyping results were obtained at the four genetic loci investigated. Sequence comparison and phylogenetic analysis showed that human-derived and animal-derived MLGs are different, and further indicated the presence of a new sub-assemblage (referred to as “AIII”), which was found exclusively in wild hoofed animals. On the other hand, there were variable levels of intra-isolate sequence heterogeneity (i.e., the presence of two overlapping nucleotide peaks at specific positions in the chromatograms, or “heterogeneous templates”) in assemblage B isolates from humans and animals, and this prevented the unambiguous identification of MLGs. Furthermore, in five human isolates and one non-human primate isolate, the assignment to assemblage B was problematic, given that one of the four markers supported an assignment to assemblage A. These findings raise concerns about the interpretation of genotyping data based on single markers, and indicate the need to understand the mechanisms that are responsible for the differences between G. duodenalis assemblages A and B.     

Structure and Evolution of the Mitochondrial Control Region of Leaf Beetles (Coleoptera: Chrysomelidae): A Hierarchical Analysis of Nucleotide Sequence Variation

Abstract To assess the levels of variation at different evolutionary scales in the mitochondrial (mt) control region of leaf beetles, we sequenced and compared the full mt control region in two genera (Chrysomela and Gonioctena), in two species within a genus (Gonioctena olivacea and G. pallida), in individuals from distant populations of these species in Europe, and in individuals from populations separated by moderate (10- to 100-km) to short (<5-km) distances. In all individuals, a highly repetitive section consisting of the tandem repetition of 12 to 17 imperfect copies of a 107- to 159-bp-long core sequence was observed. This repetitive fragment accounts for roughly 50% of the full control-region length. The sequence variability among repeated elements within the control region of a given individual depends on the species considered: the variability within any G. olivacea individual is much higher than that within G. pallida individuals. Comparisons of the repeated elements, in a phylogenetic framework, within and among individuals of G. olivacea and G. pallida suggests that the repetitive section of the control region experienced recurrent duplications/deletions, leading to some degree of concerted evolution. Comparisons between Chrysomela and Gonioctena control regions revealed virtually no significant sequence similarity, except for two long stretches of A's and several [T(T)A(A)] repeats, all found in the control region of other insect orders. Our analyses allowed us to identify portions of the control region with enough variation for population genetic or phylogeographic studies.     

The adenylate cyclase catalytic domain of Streptomyces coelicolor is carboxy-terminal

Abstract A DNA fragment of Streptomyces coelicolor encoding the carboxy-terminal catalytic domain of adenylate cyclase was cloned, sequenced and expressed in an Escherichia coli cya -defective strain where it produced nanomole levels of cAMP. The amino acid sequence of the enzyme displays similarities with the Brevibacterium liquefaciens pyruvate regulated adenylate cyclase.     

Sequence Variation in the Gpl20 region of SHIV-CN97001 during in vivo Passage

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances （divergence, diversity） were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif （GPGQ） and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.     

Purification and Chemical Characterization of Peptide G1, an Invertebrate Neuropeptide That Stimulates Cyclic GMP Metabolism

Bioassay analysis of extracts of the major neurosecretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1 (for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200-fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse-phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate-containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide approximately 60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult-inhibiting hormone) are described.     

DNA polymorphism in the conserved 190 kDa antigen gene repeat region among spotted fever group Rickettsiae

The 190 kDa protein gene of R. rickettsii (R strain) contains region of tandemly arranged repeats. Homologous sequences were discovered within the genomes of all but one species of spotted fever group Rickettsiae. Southern blots showed restriction fragment lenght polymorphisms between species of Rickettsiae indicating structural differences among repeat regions. Further analysis using polymorase chain reaction techniques indicated the repeat regions vary in size among Rickettsiae. In addition, strains of R. rickettsii that vary in virulence were found to contain polymorphisms in this region.     

Relationships among amino acid sequences of animal,microbial and plant peroxidases

Summary Relationships among 18 peroxidases amino acid sequences of animal, microbial and plant origin were examined using optimum alignment of all pairwise sequence combinations to generate a total distance matrix. The matrix was used to cluster the sequences with complete linkage (farthest neighbour) procedures. Specific distances were calculated from the total distances matrix. The patterns of specific distances for each sequence were compared to evaluate the relationships between sequences, check their significance and construct subgroups of related sequences. The results were compared with those from clustering and its resultant dendrogram; good agreement was achieved. The 18 sequences fell into two principal groups, plant peroxidases and animal/microbial peroxidases. Within the plant peroxidases four subgroups were detected; the animal/microbial peroxidases formed a fifth subgroup. Profiles were constructed for the subgroups from lists of matching amino acids generated by the alignment calculations. Superimposed lists were realigned to recognise conserved areas and elements. Individual subgroup profiles for the plant peroxidases were then combined into a single profile which in turn was combined with profiles from the animal/microbial peroxidases. The final profile suggested that numerous sequence features (motifs) were common to peroxidases of widely different function and origins.