Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Genome- and species-specific markers and genome relationships of diploid perennial species in Triticeae based on RAPD analyses.

Eight different genomes (E, H, I, P, R, St, W, and Ns) represented by 22 diploid species of the tribe Triticeae were analyzed using the random amplified polymorphic DNA (RAPD) technique. The genome relationships were obtained based on 371 RAPD fragments produced with 30 primers. The four species of the genus Psathyrostachys (having various Ns genomes) were closely related. The genomes Ee and Eb had a similarly close relationship and were distinct from all other genomes analyzed. Genomes P, R, and St were grouped in one cluster and genomes H and I in another. Genome W had a distant relationship with all other genomes. These results agree with the conclusions from studies of chromosome pairing and isozyme and DNA sequence analyses. Twenty-nine and 11 RAPD fragments are considered to be genome- and species-specific markers, respectively. One to six genome-specific markers were identified for each genome. These RAPD markers are useful in studies of genome evolution, analysis of genome composition, and genome identification.     

Synthesis of diadenosine 5',5' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.     

An alkaloid from Haplophyllum tuberculatum

A new alkaloid (+)-tuberine was isolated from Haplophyllum tuberculatum. Physicochemical and spectral evidence established its structure and stereochemistry as N- benzoyl-4′-[(2″S,3″S,6″S)-(+)-7″-acetoxy-2″-hydroxy-3″,7″-dimethyl-3″,6″-epoxyoctyloxy]phenethylamine.     

Phenological delay despite warming in wood frog Rana sylvatica reproductive timing: a 20-year study

Across all taxa, amphibians exhibit some of the strongest phenological shifts in response to climate change. As climates warm, amphibians and other animals are expected to breed earlier in response to temperature cues. However, if species use fixed cues such as daylight, their breeding timing might remain fixed, potentially creating disconnects between their life history and environmental conditions. Wood frogs Rana sylvatica are a cold-adapted species that reproduce in early spring, immediately after breeding ponds are free of ice. We used long-term surveys of wood frog oviposition timing in 64 breeding ponds over 20 yr to show that, despite experiencing a warming of 0.29°C per decade in annual temperature, wood frog breeding phenology has shifted later by 2.8 d since 2000 (1.4 d per decade; 4.8 d per °C). This counterintuitive pattern is likely the result of changes in the timing of snowpack accumulation and melting. Finally, we used relationships between climate and oviposition between 2000 and 2018 to hindcast oviposition dates from climate records to model longer-term trends since 1980. Our study indicates that species can respond to fine-grained seasonal climate heterogeneity within years that is not apparent or counterintuitive when related to annual trends across years.     

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2–5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12–90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.     

左旋千金藤啶碱D1激动—D2阻滞作用引起伏核双相放电活动的电生理研究

为确定左旋千金藤啶碱（ＳＰＤ）对中脑边缘ＤＡ神经系统的作用特性,本研究采用细胞外记录的电生理学方法,观察微电泳和尾静脉给药对６－ＯＨＤＡ损毁及未损毁大鼠的伏核（ＮＡｃ）单位放电的影响。结果显示：ＳＰＤ累积给药（０．０２－２ｍｇ／ｋｇ,ｉｖ）可诱发ＮＡｃ神经元双相放电特征,即小剂量抑制、大剂量兴奋。预先给予Ｄ２受体拮抗剂ｓｐｅｐｅｒｏｎｅ,ＳＰＤ则仅产生兴奋效应,并被Ｄ１拮抗剂ＳＣＨ－２３３９０所翻     

Nuclear volume estimation using different sampling, measurement and calculation methods

OBJECTIVE: To study the reliability of volume parameter measured on tissue sections through different sampling, measurement and calculation methods. STUDY DESIGN: The largest nuclear profile image under a 100x, NA 1.30 oil immersion objective of primary spermatocytes and spherical spermatoblasts on 11-micron-thick seminiferous tubule sections and tissue images, under a 20x objective, on 4-micron sections were captured. Their volumes were measured and calculated by the five methods provided by the Technology for Image and Graphics Engineering Research cell image analysis system. RESULTS: The nuclear volumes obtained by nucleator and area equivalent diameter on the largest nuclear profile image were almost the same, including binary images by automated and manual interactive nucleator and grey scale images only by the latter. Nuclear volumes, calculated by random Feret diameter and equivalent diameter of the perimeter, the minimal circumference of the largest nuclear profile binary image, were obviously larger than those of the nucleator and area equivalent diameter. Due to different-sized nuclear slices entrapped in the same section, those nuclear volumes from the seminiferous tubule tissue images were strikingly lower than that of the largest nuclei profile image. The shape factors of primary spermatocytes and spherical spermatoblast nuclei under 100x and 20x objectives were approximately the same. CONCLUSION: The sample preparation, sampling methods and calculation formulas suitable to nuclear form are necessary to obtain reproducible volume parameters.     

Effect of ascorbic acid on production of nitric oxide by leukocytes

The effect of ascorbic acid on suspensions of blood formic elements was studied by the ESR method. It was shown that incubation of a suspension of formic blood elements in the presence of ascorbic acid leads to the appearance of nitric oxide, which is produced by leukocytes and partially probably by thrombocytes. The formation of nitric oxide is evidenced by the appearance of nitrosyl complexes heme-NO. In this case, hemoglobin of erythrocytes serves as a natural trap for nitric oxide.