DNA sequence analysis of the defective interfering particles of bacteriophage f1.

Restriction mapping and nucleotide sequence analysis of several defective, interfering particles of bacteriophage f1 are described. These particles contain the nucleotide sequences corresponding to the carboxyl terminus of gene IV and the amino-terminus of gene II and the intergenic space between them. Tandem duplication of a portion of this intergenic space generates defective particles with novel nucleotide sequences not found in wild-type f1. This duplication is shown to contain the origin of complementary strand synthesis. Our results suggest that the duplication occurs at the site of gene II protein action, i.e. the origin of viral strand synthesis. A model is presented for the generation of these duplications in defective particles.     

Precursor structure of egg proteins in the coral Galaxea fascicularis

In the egg of the reef coral Galaxea fascicularis, four proteins (named GfEP-1 to -4) are stored in high abundance. In the present study, a cDNA containing a full-length open reading frame for GfEP-1 was cloned, and the translated protein sequence was compared to the N-terminal sequences of GfEP-2, -3, and -4. GfEP-1 and -2 were shown to be generated by processing of a precursor of 1439 amino acids, and GfEP-3 turned out to be a partial fragment of GfEP-2. The precursor protein contained regions which exhibited similarities to vitellogenins (Vgs) in bilaterian animals (oviparous vertebrates and invertebrates including nematodes, arthropods, and molluscs). This study reports the first cloning and characterization of a full-length cDNA encoding a Vg in a non-bilaterian animal, and argues that the emergence of Vg as a precursor of egg yolk proteins predated the divergence of the cnidarian and bilaterian lineages.     

AN ASSESSMENT OF CHANGES WITH TIME IN THE MARKING PATTERNS USED FOR PHOTOIDENTIFICATION OF INDIVIDUAL SPERM WHALES, PHYSETER MACROCEPHALUS

We observed changes with time in the patterns of characteristic fluke markings used to identify sperm whales. Changes were categorized as minor, moderate, or major based on their severity. These change types were found to occur at rates of 0.9%, 11.8%, and 1.3% per individual per year, respectively. Gain and loss rates for each of seven different mark types were also calculated. The highest estimated rate was the gain of small nicks at 0.08 per individual per year. Most individuals identified by us possess at least a few characteristic marks and, therefore, changes of the type observed in this study are unlikely to severely affect their recognizability. For all but one mark type, gain rates were higher than loss rates, indicating that individuals may be accumulating marks with age. Over long periods this could eventually make individuals unrecognizable, with the result that population sizes calculated from these data may be overestimated. As long as photoidentification studies are conducted sufficiently often, and these changes are as gradual as they appear to be, this problem should be minimal.     

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

The photosynthetic CO₂ fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.     

Structurally Distinct Ligands Rescue Biogenesis Defects of the KATP Channel Complex via a Converging Mechanism

Small molecules that correct protein misfolding and misprocessing defects offer a potential therapy for numerous human diseases. However, mechanisms underlying pharmacological correction of such defects, especially in heteromeric complexes with structurally diverse constituent proteins, are not well understood. Here we investigate how two chemically distinct compounds, glibenclamide and carbamazepine, correct biogenesis defects in ATP-sensitive potassium (K_ATP) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.2. We present evidence that despite structural differences, carbamazepine and glibenclamide compete for binding to K_ATP channels, and both drugs share a binding pocket in SUR1 to exert their effects. Moreover, both compounds engage Kir6.2, in particular the distal N terminus of Kir6.2, which is involved in normal channel biogenesis, for their chaperoning effects on SUR1 mutants. Conversely, both drugs can correct channel biogenesis defects caused by Kir6.2 mutations in a SUR1-dependent manner. Using an unnatural, photocross-linkable amino acid, azidophenylalanine, genetically encoded in Kir6.2, we demonstrate in living cells that both drugs promote interactions between the distal N terminus of Kir6.2 and SUR1. These findings reveal a converging pharmacological chaperoning mechanism wherein glibenclamide and carbamazepine stabilize the heteromeric subunit interface critical for channel biogenesis to overcome defective biogenesis caused by mutations in individual subunits.     

Myoblast proliferation and syncytial fusion both depend on connexin43 function in transfected skeletal muscle primary cultures

Muscles are formed by fusion of individual postmitotic myoblasts to form multinucleated syncytial myotubes. The process requires a well-coordinated transition from proliferation, through migratory alignment and cycle exit, to breakdown of apposed membranes. Connexin43 protein and cell-cycle inhibitor levels are correlated, and gap junction blockers can delay muscle regeneration, so a coordinating role for gap junctions has been proposed. Here, wild-type and dominant-negative connexin43 variants (wtCx43, dnCx43) were introduced into rat myoblasts in primary culture through pIRES-eGFP constructs that made transfected cells fluoresce. GFP-positive cells and vitally-stained nuclei were counted on successive days to reveal differences in proliferation, and myotubes were counted to reveal differences in fusion. Individual transfected cells were injected with Cascade Blue, which permeates gap junctions, mixed with FITC-dextran, which requires cytoplasmic continuity to enter neighbouring cells. Myoblasts transfected with wtCx43 showed more gap-junctional coupling than GFP-only controls, began fusion sooner as judged by the incidence of cytoplasmic coupling, and formed more myotubes. Myoblasts transfected with dnCx43 remained proliferative for longer than either GFP-only or wtCx43 myoblasts, showed less coupling, and underwent little fusion into myotubes. These results highlight the critical role of gap-junctional coupling in myotube formation.     

Correlation between cell aggregation and antibody production in IgE-producing plasma cells

Allergic conditions result in the increase of immunoglobulin (Ig)E-producing plasma cells (IgE-PCs); however, it is unclear how IgE production is qualitatively controlled. In this study, we found that IgE-PCs in spleen of immunized mice formed homotypic cell aggregates. By employing IgE-producing hybridomas (IgE-hybridomas) as a model of IgE-PCs, we showed that these cells formed aggregates in the presence of specific antigens (Ags). The formation of the Ag-induced cell aggregation involved secreted IgE and Fcγ receptor (FcγR)II/FcγRIII, but not FcεRs. Ag-induced cell aggregation plus lipopolysaccharide signaling resulted in an enhancement of IgE production in aggregated IgE-hybridomas. Furthermore, the administration of anti-FcγRII/FcγRIII antagonistic monoclonal antibody to immunized mice tended to reduce the splenic IgE-PC aggregation as well as the serum IgE levels. Taken together, our results suggested that Ag-IgE complexes induced IgE-PCs aggregation via FcγRII/FcγRIII, leading to the enhancement of IgE production. These findings suggest the presence of a novel mechanism for regulation of IgE production.     

生物多样性相关传统知识调查与评估: 以澜沧与康定两县域为例

本研究依据《县域生物多样性相关传统知识调查与评估技术规定》, 调查评估了云南省澜沧县和四川省康定市两县域生物多样性相关传统知识的分布数量与特征, 分析了传统知识保护与传承状况, 识别了主要受威胁因素和保护空缺, 旨在为我国传统知识保护和民族地区实施传统知识获取与惠益分享制度提供科学依据, 推动《生物多样性公约》与《名古屋议定书》等国际公约的履行。研究结果如下: 分别调查编目澜沧县、康定市的生物多样性相关传统知识145项、98项; 这些传统知识具有明显的地区气候与自然环境特征、生产生活实践特征、传统文化特征和地区社会经济特征; 相关部门在传统知识保护管理方面做出了积极努力, 同时包括习惯法、传统节庆等传统文化和包括祭祀、神山、神林等宗教方式在内的地方社区行动在传统知识保护与传承方面也发挥了重要作用; 生物多样性相关传统知识、创新和做法已被广泛应用于地方社区脱贫攻坚和产业发展; 传统知识保护立法仍不健全、受到威胁、保护意识薄弱及承载传统知识的生物资源减少是制约传统知识保护传承的主要因素。两县域生物多样性相关传统知识丰富, 并且地方社区有效参与了生物多样性相关传统知识的保护与可持续利用。今后应建立传统知识获取与惠益分享国家制度; 加强宣传教育, 提升保护传承意识; 因地制宜开展传统知识保护; 推动传统知识相关生物资源的保护与可持续利用。     

Comparison of the conserved region in the dnaA gene from three mollicute species

Abstract Polymerase chain reaction was carried out to amplify the conserved region (789 bp in the case of Mycoplasma capricolum ) of the dnaA gene (1350 bp in the case of M. capricolum ) of 15 representatives of the class Mollicutes using degenerate oligonucleotide primers. The dnaA gene fragments were amplified from M. mycoides subsp. capri, Spiroplasma apis and S. citri . The amino acid sequences deduced from the nucleotide sequences of the amplified fragments showed very low similarities to those of the corresponding regions of four walled bacteria. The values of similarity between any two of the three mollicute species were lower than those between any two of the four walled bacteria.