Alternative pathway activities in aged potato tuber slices measured by three methods

To find a simple and reliable oxygen electrode-based method to estimate the values of alternative pathway activity (V _alt) and its contribution to total respiration V _alt/V _t) in aged potato (Solanum tuberosum L.) tuber slices, we compared conventional hydroxamate-inhibiting method, improved hydroxamate-inhibiting method with 2,6-dichlorophenol indophenol (DCPIP), and the oxygen isotope discrimination (OID) method. The values of V _alt and V _alt/V _t obtained with an improved hydroxamate-inhibiting method with DCPIP in 12-h- and 24-h-aged slices were about twice higher than those with the conventional hydroxamate-inhibiting method. Only a relatively small difference in the values of V _alt and V _alt/V _t obtained by the OID method and the improved hydroxamate-inhibiting method with DCPIP in 12-h and 24-h-aged slices was observed. These results indicated that the improved hydroxamate-inhibiting method with DCPIP could be considered as a new, simple, and reliable technique for the noninvasive assay of the AP activity.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 311–315.Original English Text Copyright © 2005 by Hou, Zhou, Kong, Liang, Zhang.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Glycosidic Bond Conformation Preference Plays a Pivotal Role in Catalysis of RNA Pseudouridylation: A Combined Simulation and Structural Study

The most abundant chemical modification on RNA is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.     

The regulatory mechanism of extracellular Hsp90{alpha} on matrix metalloproteinase-2 processing and tumor angiogenesis

Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone that is essential for eukaryotic homeostasis. Hsp90α can also be secreted extracellularly, where it has been shown to be involved in tumor metastasis. Extracellular Hsp90α interacts with and promotes the proteolytic activity of matrix metalloproteinase-2 (MMP-2). However, the regulatory mechanism of Hsp90α on MMP-2 activity is still unknown. Here we show that Hsp90α stabilizes MMP-2 and protects it from degradation in tumor cells. Further investigation reveals that this stabilization effect is isoform-specific, ATP-independent, and mediated by the interaction between the Hsp90α middle domain and the MMP-2 C-terminal hemopexin domain. Moreover, this mechanism also applies to endothelial cells that secrete more Hsp90α in their proliferating status. Furthermore, endothelial cell transmigration, Matrigel plug, and tumor angiogenesis assays demonstrate that extracellular Hsp90α promotes angiogenesis in an MMP-2-dependent manner. In sum, this study provides new insights into the molecular mechanism of how Hsp90α regulates its extracellular client proteins and also reveals for the first time the function of extracellular Hsp90α in promoting tumor angiogenesis.     

Silkworm coatomers and their role in tube expansion of posterior silkgland

Background

Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.

Methodology/Principal Findings

Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion.

Conclusions/Significance

The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.     

Characteristics of sun- and shade-adapted populations of an endangered plant <Emphasis Type="Italic">Primulina tabacum</Emphasis> Hance

Primulina tabacum Hance is an endangered perennial herb distributed in calcium-rich and nitrogen-limited soil of the karst limestone areas in southern China. The morphological, ultrastructural, and physiological traits were determined for P. tabacum populations growing in three different environment conditions: twilight zone of a cave (site TZ, extremely low light intensity), at a cave entrance (site EZ, low light intensity), and in an open area (site OA, high light intensity). At site OA, P. tabacum plants were exposed to high light (635 μmol m⁻² s⁻¹ of mean daily photosynthetically active radiation) with drought stress, and expressed traits to minimize light capture and water loss. Compared to plants at sites EZ and TZ, those at site OA had thicker leaves with higher densities of stomata and pubescence, higher palisade/spongy ratio, higher light-saturated rate of net photosynthetic rate (P _max), higher biomass, higher non-photochemical quenching coefficient (NPQ), and higher light saturation point (LSP) but fewer grana per chloroplast and less thylakoid stacking per granum. In contrast, P. tabacum growing at the cave vicinities: EZ (mean daily irradiance 59 μmol m⁻² s⁻¹) and TZ (mean daily irradiance 11 μmol m⁻² s⁻¹) showed typical shade-adapted characteristics for optimum light capture. The presence of sun- and shade-adapted characteristics indicates that P. tabacum has different strategies to cope with different environments but whether these strategies reflect genetic selection or phenological plasticity is yet to be determined. Such variability in physiological and morphological traits is important for the survival of P. tabacum in heterogeneous light conditions.     

EPSVR and EPMeta: prediction of antigenic epitopes using support vector regression and multiple server results

Background  

Accurate prediction of antigenic epitopes is important for immunologic research and medical applications, but it is still an open problem in bioinformatics. The case for discontinuous epitopes is even worse - currently there are only a few discontinuous epitope prediction servers available, though discontinuous peptides constitute the majority of all B-cell antigenic epitopes. The small number of structures for antigen-antibody complexes limits the development of reliable discontinuous epitope prediction methods and an unbiased benchmark to evaluate developed methods.     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.     

Characterization of the zebrafish vascular endothelial growth factor A gene: comparison with vegf-A genes in mammals and Fugu

Vascular endothelial growth factor (VEGF-A) is a key angiogenic growth factor which regulates vertebrate embryonic vascularization, adult physiology such as wound healing and reproduction as well as many human diseases. To understand the evolution and regulation of this gene in vertebrates, we have isolated and characterized the zebrafish vegf-A gene and compared it with VEGF-A genes of human, mouse as well as an in silico isolated VEGF-A homologue from pufferfish. Our results indicate that the zebrafish vegf-A gene is organized similarly to mammalian and Fugu VEGF-A genes, with eight exons interrupted by seven introns. However, zebrafish vegf-A introns are generally larger than mammalian introns while Fugu VEGF-A introns are much smaller. Furthermore, zebrafish exon 6 (z6) has a unique sequence while Fugu's exon 6 is highly homologous to the mammalian counterparts. Alternative splicing generates multiple vegf-A mRNA isoforms in zebrafish with Vegf(121) as the dominant isoform in adult and Vegf(165) as the dominant isoform in early embryos. The exon z6 containing isoform Vegf(12345z678) is only detected in heart, muscle, and early embryos while another isoform Vegf-A(1234577)(a)(8) is only detected in heart. Furthermore, no conserved 5' flanking sequences between zebrafish and Fugu were observed while numerous conserved regions exist between human and mouse in this area. These results suggest both conserved and diverged functions of VEGF-A from fish to mammals since the separation of these two groups from their common ancestor about 450 million years ago and a diverged regulation of this gene since the separation of zebrafish from Fugu. These data will be valuable for future studies of VEGF-A gene regulation and function in different vertebrates.     

Influence of chk1 and plk1 silencing on radiation- or cisplatin-induced cytotoxicity in human malignant cells

The G2/M checkpoint is an attractive pathway for targeting and sensitizing tumor cells to cancer treatment. Abrogation of the G2/M checkpoint by targeting molecules, such as checkpoint kinase 1 (chk1), increases DNA breakage and sensitizes tumor cells to anti-tumoral agents. However, most of the previously described G2/M abrogators are actually targeting the G2-M border checkpoints rather than mitotic checkpoints. This prompted us to test the effects of combined targeting of chk1 and a critical regulator of mitosis, polo-like kinase 1 (plk1). Chk1 and plk1 were found to be co-expressed in 70% of primary neoplastic tissues we examined. Asynchronized tumor cells were treated with different DNA damaging-agents to activate G1/S, S or G2/M checkpoints. Either chk1 or plk1-specific antisense oligodeoxynucleotides (ASODN) enhanced DNA damaging agent-induced apoptosis. When used in combination, however, chk1- plus plk1-specific ASODN failed to produce synergistic effects. Moreover, selective targeting of plk1 or chk1 in tumor xenografts of mice by oncolytic adenovirus mutants demonstrated potent anti-tumoral efficacy in the presence of low dose cisplatin. Again, combined targeting of chk1 and plk1 did not further enhance anti-tumoral efficacy. We concluded that combined targeting of chk1 and plk1 was not superior to either targeting chk1 or plk1 alone, which suggested that chk1 and plk1 silencing might overlap in their mechanism of action. Whether combined targeting of chk1 with other, more specific mitotic regulators would synergistically sensitize tumor to anti-neoplastic therapeutics needs to be further clarified. Qinglei Gao and Xiaoyuan Huang contributed equally to this work.