Perspective: Time scales in scientific research with an emphasis on microbial cellular and molecular research

Scientists use time to describe and research the universe in which humans live. Geologists and evolutionary biologists often use time scales in the millions to billions of years while biochemists and molecular biologists use time scales in the milliseconds or less. The atom smashers use time scales that are almost the speed of light. However, in some areas of research such as molecular-based activities in cells, it is very challenging to obtain data sets in molecular time scales. This has been a challenge to obtaining accurate and precise measurements at the cell and molecular levels of organization in living organisms. Measurements of specific cellular and molecular activities are often made over time scales longer than the actual molecular events. The data sets obtained become estimates over seconds, minutes and hours and not measurements over milli- and nanoseconds. The question can then be posed — how representative and accurate are our data sets when the time scales are not synchronized with the actual living events? In this article, the role of time scales in scientific research and our understanding of living microorganisms are examined with an emphasis on cell and molecular time scales.     

Measuring the relationship between dietary quality and body size in primates

The feeding niche and the body size of any species are fundamental parameters that constrain the evolution of many other phenotypic characters. Moreover, previous work has shown that body size and diet are correlated, as a consequence of the negative allometry of metabolic rate. Unfortunately, the precise form of the association between body size and diet has never been specified, principally because no suitable cross-species measure of diet has been advanced. Here we develop a measure of diet that is sensitive over the whole spectrum of primate feeding niches, and use this measure to define the relationship between body size and diet for a sample of 72 primate species. Subsequently, we present several examples of how behavioral and ecological hypotheses can be tested by examining the extent to which particular species deviate from the general diet-body size pattern.     

肝囊型包虫病非手术治疗方法的研究进展

肝囊型包虫病(简称肝包虫病)是一种由细粒棘球蚴感染肝脏引起的人兽共患寄生虫病,严重危害人类以及家畜的健康。近年来,由于该病术后复发率较高且易出现并发症,非手术治疗方法如介入技术、放射治疗以及药物治疗均得到了不断的发展。介入技术对于肝包虫病的治疗是一种比较新颖的方法,其临床应用时间较短,主要包括经皮穿刺治疗、射频消融、门静脉栓塞,以经皮穿刺治疗最为常见。放射治疗肝包虫病尚处于研究阶段,效果尚不完全明确。苯并咪唑类药物及其新剂型、非苯并咪唑类药物如地塞米松等及中草药治疗肝包虫病也均具有各自的优缺点。本文主要对上述非手术治疗方法及优缺点进行了综述。     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.     

Modulation of SCF��-TrCP-dependent I��B�� Ubiquitination by Hydrogen Peroxide

Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-κB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H₂O₂) have anti-inflammatory effects in neutrophils, including inhibition of the degradation of IκBα after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H₂O₂ resulted in diminished ubiquitination of IκBα and decreased SCF^β-TrCP ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H₂O₂ was associated with reduced binding between phosphorylated IκBα and SCF^β-TrCP but no change in the composition of the SCF^β-TrCP complex. Lipopolysaccharide-induced SCF^β-TrCP ubiquitin ligase activity as well as binding of β-TrCP to phosphorylated IκBα was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H₂O₂ are increased. Exposure to H₂O₂ resulted in oxidative modification of cysteine residues in β-TrCP. Cysteine 308 in Blade 1 of the β-TrCP β-propeller region was found to be required for maximal binding between β-TrCP and phosphorylated IκBα. These findings suggest that the anti-inflammatory effects of H₂O₂ may result from its ability to decrease ubiquitination as well as subsequent degradation of IκBα through inhibiting the association between IκBα and SCF^β-TrCP.     

In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots

Changes in membrane morphology and membrane protein dynamics based on its fluidity are critical for cancer metastasis. However, this subject has remained unclear, because the spatial precision of previous in vivo imaging has been limited to the micrometer level and single molecule imaging is impossible. Here, we have imaged the membrane dynamics of tumor cells in mice with a spatial precision of 7–9 nm under a confocal microscope. A metastasis-promoting factor on the cell membrane, protease-activated receptor 1 (PAR1), was labeled with quantum dots conjugated with an anti-PAR1 antibody. Movements of cancer cells and PAR1 during metastasis were clearly observed in vivo. Images used to assess PAR1 dynamics were taken of representative cells for four stages of metastasis; i.e. cancer cells far from blood vessels in tumor, near the vessel, in the bloodstream, and adherent to the inner vascular surface in the normal tissues near tumor were photographed. The diffusion constant of PAR1 in static cells far from tumor blood vessels was smaller than in moving cells near the vessels and in the bloodstream. The diffusion constant of cells adhering to the inner vascular surface in the normal tissues was also very small. Cells formed membrane protrusion during migration. The PAR1 diffusion constant on these pseudopodia was greater than in other membrane regions in the same cell. Thus, the dynamics of PAR1 movement showed that membrane fluidity increases during intravasation, reaches a peak in the vessel, decreases during extravasation, and is also higher at locally formed pseudopodia.     

A comparison of hole punch and needle punch methods for the measurement of seagrass productivity

Seagrass productivity, as leaf extension, was measured using the hole punch and needle punch techniques. These methods have been widely implemented to determine seagrass leaf extension rates, yet there is no evidence in the literature of a comparison between methods. The hole punch method involves removing part of the basal area of a seagrass leaf and it was proposed that this measurement technique may affect the leaf extension rates that are being measured. Leaf extension rates were measured in Posidonia sinuosa meadows off the coast of Perth, Western Australia. There were no significant differences in seagrass leaf extension between the two methods. The hole punch method is favoured, as measurement of incremental leaf growth is facilitated by the obvious hole left by the punch. The needle punch method leaves lesions on seagrass leaves that are easily confused with other lesions, possibly left by invertebrate grazers. These findings are likely to be applicable to other straplike seagrass species, though a similar comparison is recommended in the initial stages of a study.     

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.