[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1

Summary The synthesis of [2-³H]ATP with specific activity high enough to use for ³H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single ³H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg²⁺, S1 converts [2-³H]ATP to [2-³H]ADP and the complex S1.Mg[2-³H]ADP has ADP bound in the active site. At 0°C, 1D ³H NMR spectra of S1.Mg[2-³H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-³H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-³H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (V_i) to produce S1.Mg[2-³H]ADP.V_i shifts both peaks slightly more upfield without chaning their widths or relative areas.     

海南潜在世界自然遗产地的突出普遍价值初探

世界自然遗产地是全球最具有保护价值的自然保护地,强调全球突出普遍价值的完整性和在全球的唯一性。世界自然遗产有助于更好地保护生态系统的完整性和原真性,促进人类与自然的可持续发展。该研究在大量文献资料的基础上,以海南潜在世界自然遗产地(海南热带雨林国家公园)原生动植物及植被群落(亚洲北缘热带雨林)为研究对象,从植被类型、物种多样性、区系组成、特有种等生物生态过程方面,评估海南潜在世界自然遗产地的全球突出普遍价值。结果表明:(1)海南潜在世界自然遗产地分布有3 653种野生维管植物,资源植物种类丰富。陆栖脊椎动物有540种,各类野生动物占全国的比例高达10%～30%,生物多样性极高。(2)植物区系独特,海南岛的热带雨林植被区划属于印度-马来雨林群系,属马来区的部分呈现出热带性和与中国华南大陆的共源性显示出明显的热带边缘性质,为中国华南植物区系与亚洲热带雨林的过渡类型。(3)植物区系中的植物物种特有性较低,特有属仅有7个,特有种仅约占岛内植物的1/10,较低的特有性表明了其大陆起源特征,是生物多样性不可替代的元素,具有鲜明的环境指示特色。该研究明确了海南潜在世界自然遗产地在全球背景下的突出普遍价值,为海南未来申遗提供科学依据和技术支撑。     

Cleavage of double-stranded DNA by manganese tris(methylpyridiniumyl)porphyrin linked to 3′-spermine oligonucleotides

 Cleavage of double-stranded DNA was performed with cationic manganese porphyrin complexes linked via a spermine tether to the 3′- or 5′-side of triple-helix-forming oligonucleotides (cleaver-TFO conjugates). The targeted sequence was a 15-polypurine sequence present in the env gene of HIV-1 (positions 7301–7315). The presently used TFOs contain only thymine and 5-methylcytosine residues and one adenine at the 3′-end in order to be able to easily introduce a 3′-polyamine linker by reductive amination of the corresponding 3′-apurinic polypyrimidine oligonucleotides. With this method we prepared these TFO-cleaver conjugates in 45% yield with only two equivalents of the Mn-TrisMPyP-COOH precursor. These new metalloporphyrin-TFO conjugates were able to cleave a complementary 45-mer duplex at 10 nM concentration with only ten equivalents of TFO-cleaver. Conjugates without spermine, without 5-methylcytosine, with a random sequence or with the managanese porphyrin-spermine entity on the 5′-end of TFOs were synthesized for comparative studies. Received: 6 December 1995 / Accepted: 5 February 1996     

A model for the mechanism of precise integration of a microinjected transgene

A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5 and 3 joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5 end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the E gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.     

Scarification,fertilization and herbicide treatment effects on planted conifers and soil fertility

The influences of soil surface modification (blade scarification and plastic mulching), fertilization and herbicide application on soil nutrient and organic carbon content and tree growth and foliar nutrient status were examined after seven years in a study located within the Great Lakes-St. Lawrence forest region of Canada. Plots had been planted with white pine (Pinus strobus L.) and white spruce (Picea glauca [Moench] Voss) seedlings. Light (PAR), soil moisture and temperature were monitored and recorded throughout the growing season. Forest floor and soil mineral (0–20 cm layer) samples were collected from all experimental plots, except those which had plastic mulching. Foliar samples were collected in autumn and analysed for N, P and K and storage compounds. Seedling mortality was 20% higher in unscarified plots. Combined silvicultural treatments increased productivity as much as 14 times, but scarification reduced soil carbon and nutrient capital 2–3 fold. Herbicide application reduced soil carbon by at least 20 %. Foliar nutrient, protein, starch and lipid contents in autumn were little affected by treatment. The future management of such stands in Canada probably will include more shelterwood harvesting and crop rotations, silvicultural systems that are more closely aligned with natural forest succession.     

Towards an understanding of the relations between tree nutrition,nutrient cycling and environment

The paper contains a discussion of the interrelations between the sciences used by managers of forest land to improve their management, in particular with respect to the plant nutrient economy of the forest ecosystems. Both site studies and studies of nutrient cycling have been carried out for long periods without proper consideration of tree nutrition. Therefore these studies contributed less to the understanding of the role of nutrients as regulators of processes in ecosystems than might have been expected. This situation has improved, especially within the last decade. In addition the necessity to manage forest land for environmental values as well as for forest yield requires new interdisciplinary approaches in the study of the roles of plant nutrients in the forest. Even more branches of biological and environmental sciences than those just mentioned must be involved.     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

What studies of turbellarian embryos can tell us about the evolution of development mechanisms

In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed.     

Functional domains of the gonadotropin-releasing hormone receptor

Summary 1. The cloning of the mammalian gonadotropin-releasing hormone receptor sets the stage for rapid progress in understanding the structure of the receptor, its interaction with ligand, and its mechanisms of activation.2. The receptor is a 327 to 328-amino acid seven-transmembrane domain G protein-coupled receptor.3. Recent site-direct mutagenesis studies have provided considerable insight into glycosylation of the receptor, the arrangement of the helices, and the ligand binding domains.