An anatomical study of anther development in Acorus L.: Phylogenetic implications

 Critical morphological synapomorphies have not been found in support of the Acoranan hypothesis, the molecular phylogenetic discovery that Acoranae are the basal monocots. The previously undetermined pattern of anther wall development in Acorus has been suggested to be one such character. Two main types of anther wall development have been recognized: 1) the “monocotyledonous” type, which characterizes both monocots and dicots, and 2) the “dicotyledonous” type, which is almost exclusively found among dicots. An anatomical study of anther wall development in Acorus was here undertaken using the electron microscope. Development of the anther wall in Acorus was found to be somewhat irregular or perhaps even intermediate between the two types although largely consistent with the “monocotyledonous” type. The presumed significance of anther wall development and other critical morphological characters to the Acoranan hypothesis in the absence of knowledge about the sister group to the monocots is evaluated. Received August 28, 2000 Accepted February 19, 2001     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology

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Highlights

• •Human spermatozoa possess cells of poor morphology that lack nuclear integrity.
• •These cells can be isolated by density separation.
• •Mass spectrometry reveals their nuclei contain excess protein.
• •TOP2A is a promising marker of this poor nuclear development.

     

First report of a green toad (Bufo viridis sensu lato) in the Early Pleistocene of Spain: Palaeobiogeographical and palaeoecological implications

For the first time unequivocal fossil remains of a green toad (Bufo viridis s.l.) are described in the Iberian Peninsula. The fossils come from the Cueva Victoria site, a late Early Pleistocene (ca. 1.1–1.2 Ma) karstic filling in semi-arid southeastern Spain (Murcia region). By extension, other remains from two other Early Pleistocene Spanish localities, Barranco León D (ca. 1.3 Ma) and Almenara-Casablanca 3 (ca. 1.1 Ma), are cautiously attributed to the group B. viridis. The B. viridis group was previously reported with some uncertainty to the west of its current distribution area in Western Europe (Spain and France) in the Pliocene (Bufo cf. viridis) and less probably in the Early Miocene (Bufo aff. viridis). Since no osteological differences have been established between the recently described extant species of B. viridis s.l. (e.g. Bufo balearicus, Bufo siculus, Bufo boulengeri, B. viridis sensu stricto and Bufo variabilis) no precise palaeobiogeographical relationships can be drawn for the Spanish fossils. However, the occurrence of a third species of bufonid toad during the Pleistocene in the South of the Iberian Peninsula raises some interesting ecological questions in relation to the local disappearance of the green toad, which can be hypothetically linked to the intensification of the Pleistocene glacial/interglacial climate dynamic or to probable competition with another toad, Bufo calamita.     

Hepatic DNAJB9 Drives Anabolic Biasing to Reduce Steatosis and Obesity

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Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Morphological and Molecular Identification of Spirometra Tapeworms (Cestoda: Diphyllobothriidae) from Carnivorous Mammals in the Serengeti and Selous Ecosystems of Tanzania

Spirometra tapeworms (Cestoda: Diphyllobothriidae) collected from carnivorous mammals in Tanzania were identified by the DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and internal transcribed spacer 1 (ITS1), and by morphological characteristics. A total of 15 adult worms were collected from stool samples and carcasses of Panthera leo, Panthera pardus, and Crocuta crocuta in the Serengeti and Selous ecosystems of Tanzania. Three Spirometra species: S. theileri, S. ranarum and S. erinaceieuropaei were identified based on morphological features. Partial cox1 sequences (400 bp) of 10 specimens were revealed. Eight specimens showed 99.5% similarity with Spirometra theileri ({"type":"entrez-nucleotide","attrs":{"text":"MK955901","term_id":"1796114604","term_text":"MK955901"}}MK955901), 1 specimen showed 99.5% similarity with the Korean S. erinaceieuropaei and 1 specimen had 99.5% similarity with Myanmar S. ranarum. Sequence homology estimates for the ITS1 region of S. theileri were 89.8% with S. erinaceieuropaei, 82.5% with S. decipiens, and 78.3% with S. ranarum; and 94.4% homology was observed between S. decipiens and S. ranarum. Phylogenetic analyses were performed with 4 species of Spirometra and 2 species of Dibothriocephalus (=Diphyllobothrium). By both ML and BI methods, cox1 and ITS1 gave well supported, congruent trees topology of S. erinaceieuropaei and S. theileri with S. decipiens and S. ranarum forming a clade. The Dibothriocephalus species were sisters of each other and collectively forming successive outgroups. Our findings confirmed that 3 Spirometra species (S. theileri, S. ranarum, and S. erinaceieuropaei) are distributed in the Serengeti and Selous ecosystems of Tanzania.     

Long noncoding RNA PTENP1 affects the recovery of spinal cord injury by regulating the expression of miR-19b and miR-21

Exosomes derived from differentiated P12 cells and MSCs were proved to suppress apoptosis of neuron cells, and phosphatase and tensin homolog pseudogene 1 (PTENP1) was reported to inhibit cell proliferation. In this study, we aimed to investigate the role of PTENP1 in the process of post-spinal cord injury (SCI) recovery, so as to evaluate the therapeutic effects of exosomes derived from MSCs transfected with PTENP1 short hairpin RNA (shRNA), as a type of novel biomarkers in the treatment of SCI. Electron microscopy was used to observe the morphology of different exosomes. Real-time polymerase chain reaction and western blot, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry, Nissl staining, immunohistochemistry assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were conducted to investigate and validate the underlying molecular signaling pathway. PTENP1-shRNA downregulated PTENP1 and PTEN while upregulating miR-21 and miR-19b. PTENP1-shRNA also accelerated cell apoptosis and reduced cell viability. In addition, PTENP1 reduced the miR-21 and miR-19b expression by directly targeting miR-21 and miR-19b. Meanwhile, both miR-21 and miR-19b reduced the expression of PTEN by directly targeting the 3′-untranslated region of PTEN. Furthermore, PTEN level and apoptosis index of neuron cells was the highest in the SCI group, while the treatment with exosomes+PTENP1-shRNA reduced the PTEN expression to a level similar to that in the sham group. Finally, PTENP1 inhibited miR-21 and miR-19b expression but upregulated PTEN expression. The upregulation of miR-21/miR-19b also suppressed the apoptosis of neuron cells by downregulating the PTEN expression. PTENP1 is involved in the recovery of SCI by regulating the expression of miR-19b and miR-21, and exosomes from PTENP1-shRNA-transfected cells may be used as a novel biomarker in SCI treatment.