Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor.

We have previously identified a Thr- and Cys-rich thermal hysteresis (antifreeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times the freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was undertaken for other antifreeze types. Many active proteins were observed, but all appeared to be isoforms of the THP that differed in their number of 12-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitutions, and N-linked glycosylation. Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tightly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated with the need to rapidly increase gene product in response to climate change. A three-dimensional model is proposed for the fully disulfide-bonded structure of T. molitor THP, which can accommodate addition or deletion of 12-amino acid repeats. The structure is a beta-helix that places most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.     

桔梗生物学的研究

桔梗既是一种资源植物;又为常用中药,可宣肺祛痰、利咽排脓;并供作菜肴,别具风味,颇受欢迎。不论药用或食用,多年来皆依靠采挖野生资源。近年来,由于荒原减少,再加上只采不育,野生资源日益枯竭,既使过去每年调出量很大的黑龙江省亦感紧缺!为此,我们自1981年以来,对桔梗的生物学进行了一些研究,希望对保护和发展这一资源,乃至引种栽培提供理论依据。     

东北蒿属一新种

本文发表了产于东北的蒿属一新种,即肇东蒿Artemisia zhao-dongensis G.Y.Chang et M.Y.Liu。     

The Microtubule-associated Protein EB1 Links AIM2 Inflammasomes with Autophagy-dependent Secretion

Inflammasomes are multi-protein complexes that regulate chronic inflammation-associated diseases by inducing interleukin-1 β (IL-1β) secretion. Numerous components involved in inflammasome activation have been identified, but the mechanisms of inflammasome-mediated IL-1β secretion have not yet been fully explored. Here, we demonstrate that end-binding protein 1 (EB1), which is required for activation of AIM2 inflammasome complex, links the AIM2 inflammasome to autophagy-dependent secretion. Imaging studies revealed that AIM2 inflammasomes colocalize with microtubule organizing centers and autophagosomes. Biochemical analyses showed that poly(dA-dT)-activated AIM2 inflammasomes induce autophagy and IL-1β secretion in an LC3-dependent fashion. Furthermore, depletion of EB1 decreases autophagic shedding and intracellular trafficking. Finally, we found that the 5′-AMP activated protein kinase may regulate this EB1-mediated autophagy-based inflammasome-induced secretion of IL-1β. These findings reveal a novel EB1-mediated pathway for the secretion of IL-1β.     

Substrate Compositional Variation with Tissue/Region and Gba1 Mutations in Mouse Models–Implications for Gaucher Disease

Gaucher disease results from GBA1 mutations that lead to defective acid β-glucosidase (GCase) mediated cleavage of glucosylceramide (GC) and glucosylsphingosine as well as heterogeneous manifestations in the viscera and CNS. The mutation, tissue, and age-dependent accumulations of different GC species were characterized in mice with Gba1 missense mutations alone or in combination with isolated saposin C deficiency (C*). Gba1 heteroallelism for D409V and null alleles (9V/null) led to GC excesses primarily in the visceral tissues with preferential accumulations of lung GC24∶0, but not in liver, spleen, or brain. Age-dependent increases of different GC species were observed. The combined saposin C deficiency (C*) with V394L homozygosity (4L;C*) showed major GC18∶0 degradation defects in the brain, whereas the analogous mice with D409H homozygosity and C* (9H;C*) led to all GC species accumulating in visceral tissues. Glucosylsphingosine was poorly degraded in brain by V394L and D409H GCases and in visceral tissues by D409V GCase. The neonatal lethal N370S/N370S genotype had insignificant substrate accumulations in any tissue. These results demonstrate age, organ, and mutation-specific quantitative differences in GC species and glucosylsphingosine accumulations that can have influence in the tissue/regional expression of Gaucher disease phenotypes.     

Critical role of WW domain phosphorylation in regulating phosphoserine binding activity and Pin1 function.

Phosphoserine-binding modules help determine the specificity of signal transduction events. One such module, the group IV WW domain, plays an essential role in targeting the phosphorylation-specific prolyl isomerase Pin1 to its substrates. These modules require Ser/Thr phosphorylation of their ligands for binding activity. However, phosphorylation of these modules and its functional significance have not been described, nor is it known whether the function of Pin1 is regulated. Here we show that Pin1 WW domain is phosphorylated on Ser(16) both in vitro and in vivo. Further, this phosphorylation regulates the ability of the WW domain to mediate Pin1 substrate interaction and cellular localization. Moreover, both Pin1 and WW domain mutants refractory to Ser(16) phosphorylation act as dominant-negative mutants to induce mitotic block and apoptosis and increase multinucleated cells with 8 N DNA content. Thus, phosphorylation is a new mechanism critical for regulating WW domain phosphoserine binding activity and Pin1 function.     

HURP regulates chromosome congression by modulating kinesin Kif18A function

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics [1-4]. The?dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule-associated proteins (MAPs) [4-9]. Recently, a MAP known as HURP (hepatoma upregulated protein) was identified [10-12]. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP) releases HURP from the importin β inhibitory complex and allows it to localize to the kinetochore fiber (k-fiber) [12, 13]. HURP stabilizes k-fibers and promotes chromosome congression [12, 14, 15]. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our results demonstrate in part the regulatory mechanism for Kif18A during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.