Comparative genomics of four Liliales families inferred from the complete chloroplast genome sequence of Veratrum patulum O. Loes. (Melanthiaceae)

The sequence of the chloroplast genome, which is inherited maternally, contains useful information for many scientific fields such as plant systematics, biogeography and biotechnology because its characteristics are highly conserved among species. There is an increase in chloroplast genomes of angiosperms that have been sequenced in recent years. In this study, the nucleotide sequence of the chloroplast genome (cpDNA) of Veratrum patulum Loes. (Melanthiaceae, Liliales) was analyzed completely. The circular double-stranded DNA of 153,699 bp consists of two inverted repeat (IR) regions of 26,360 bp each, a large single copy of 83,372 bp, and a small single copy of 17,607 bp. This plastome contains 81 protein-coding genes, 30 distinct tRNA and four genes of rRNA. In addition, there are six hypothetical coding regions (ycf1, ycf2, ycf3, ycf4, ycf15 and ycf68) and two open reading frames (ORF42 and ORF56), which are also found in the chloroplast genomes of the other species. The gene orders and gene contents of the V. patulum plastid genome are similar to that of Smilax china, Lilium longiflorum and Alstroemeria aurea, members of the Smilacaceae, Liliaceae and Alstroemeriaceae (Liliales), respectively. However, the loss rps16 exon 2 in V. patulum results in the difference in the large single copy regions in comparison with other species. The base substitution rate is quite similar among genes of these species. Additionally, the base substitution rate of inverted repeat region was smaller than that of single copy regions in all observed species of Liliales. The IR regions were expanded to trnH_GUG in V. patulum, a part of rps19 in L. longiflorum and A. aurea, and whole sequence of rps19 in S. china. Furthermore, the IGS lengths of rbcL-accD-psaI region were variable among Liliales species, suggesting that this region might be a hotspot of indel events and the informative site for phylogenetic studies in Liliales. In general, the whole chloroplast genome of V. patulum, a potential medicinal plant, will contribute to research on the genetic applications of this genus.     

Mirror‐symmetric microtubule assembly and cell interactions drive lumen formation in the zebrafish neural rod

By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror‐symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.     

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5–188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.     

Regulation of cell proliferation and migration in gallbladder cancer by zinc finger X-chromosomal protein

Gallbladder carcinoma (GBC) is one of the mostly aggressive and fatal malignancies. However, little is known about the oncogenic genes that contributed to the development of GBC. Zinc finger X-chromosomal protein (ZFX) was a novel member of the Krueppel C2H2-type zinc-finger protein family and its down-regulation led to impaired cell growth in human laryngeal squamous cell carcinoma. Here, we aim to investigate the function of ZFX in GBC cell proliferation and migration. Loss of function analysis was performed on GBC cell line (GBC-SD) using lentivirus-mediated siRNA against ZFX. The proliferation, in vitro tumorigenesis (colony-formation) ability as well as cell migration was significantly suppressed after GBC-SD cells which were infected with ZFX-siRNA-expressing lentivirus (Lv-shZFX). Our finding suggested that ZFX promoted the growth and migration of GBC cells and could present a potential molecular target for gene therapy of GBC.     

Multivariate palaeoecological analysis of a late quaternary small mammal succession from North Bulgaria

The basic assumption of the analysis is that the stratigraphical change of relative abundance of the small mammal species is a phenomenon due to a certain number of environmental factors. In order to reveal these factors and to make some palaeoclimatic reconstructions the following approaches have been used: 1. Analysis of intrinsic structure of the initial data by using principal component analysis (PCA)—delimitation of recurrent groups of species and ordination of assemblages; 2. Extension backward in time of known ecological preference of taxa, forming a particular recurrent group; 3. Comparison of the results with the analysis of assemblages from “known”; (Holocene) environment. On the basis of ecological appearance of particular recurrent groups it is assumed that the main factors are temperatures and effective moisture. The reconstructed values for these environmental factors obtained on the basis of the Holocene recurrent groups of small mammals shows similarity to the recent climate in the area.     

Small Bowel and Plasma Gastrin Rhythms in Cats

The purpose of this experiment was to study the possible role of the gastric antrum and small bowel in the rhythm(s) of plasma gastrin. The cat was used as the laboratory animal. Three groups of cats were provided with a gastric fistula for the study of gastric acid and plasma gastrin rhythms. The first group (N = 7) served as controls. A second group (N = 3) was antrectomized and later subjected to a 80% small bowel resection. Gastric acid secretions were collected every 30 min from 0800 to 2400. Blood samples for determination of gastrin were drawn every 2hr from 0800 to 2400. In control animals a circadian (i.e.<24hr) and 3 ultradian (i.e.<24 hr) rhythms were detected for acid output. In the antrectomized cats, circadian and ultradian rhythms were documented. After small bowel resection circadian and ultradian rhythms in gastric acid secretion were observed. For plasma gastrin, circadian and ultradian rhythms were found in the control cats. In the antrectomized cats no rhythms were observed. After small bowel resection an ultradian rhythm reappeared in these antrectomized cats. Removal of the antrum in the cat induces disappearance of circadian and ultradian rhythms of plasma gastrin but fails to modify the acid rhythms. Small bowel resection results in the reappearance of an ultradian rhythm for plasma gastrin and a shift in acrophase for the circadian rhythm in acid secretion.     

α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity

α-Synuclein is an abundant presynaptic protein and a primary component of Lewy bodies in Parkinson disease. Although its pathogenic role remains unclear, in healthy nerve terminals α-synuclein undergoes a cycle of membrane binding and dissociation. An α-synuclein binding assay was used to screen for vesicle proteins involved in α-synuclein membrane interactions and showed that antibodies directed to the Ras-related GTPase Rab3a and its chaperone RabGDI abrogated α-synuclein membrane binding. Biochemical analyses, including density gradient sedimentation and co-immunoprecipitation, suggested that α-synuclein interacts with membrane-associated GTP-bound Rab3a but not to cytosolic GDP-Rab3a. Accumulation of membrane-bound α-synuclein was induced by the expression of a GTPase-deficient Rab3a mutant, by a dominant-negative GDP dissociation inhibitor mutant unable to recycle Rab3a off membranes, and by Hsp90 inhibitors, radicicol and geldanamycin, which are known to inhibit Rab3a dissociation from membranes. Thus, all treatments that inhibited Rab3a recycling also increased α-synuclein sequestration on intracellular membranes. Our results suggest that membrane-bound GTP-Rab3a stabilizes α-synuclein on synaptic vesicles and that the GDP dissociation inhibitor·Hsp90 complex that controls Rab3a membrane dissociation also regulates α-synuclein dissociation during synaptic activity.     

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.