Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell–Giant Unilamellar Vesicle Electrofusion

Here, we report a method for introducing large objects of up to a micrometer in diameter into cultured mammalian cells by electrofusion of giant unilamellar vesicles. We prepared GUVs containing various artificial objects using a water-in-oil (w/o) emulsion centrifugation method. GUVs and dispersed HeLa cells were exposed to an alternating current (AC) field to induce a linear cell–GUV alignment, and then a direct current (DC) pulse was applied to facilitate transient electrofusion. With uniformly sized fluorescent beads as size indexes, we successfully and efficiently introduced beads of 1 µm in diameter into living cells along with a plasmid mammalian expression vector. Our electrofusion did not affect cell viability. After the electrofusion, cells proliferated normally until confluence was reached, and the introduced fluorescent beads were inherited during cell division. Analysis by both confocal microscopy and flow cytometry supported these findings. As an alternative approach, we also introduced a designed nanostructure (DNA origami) into live cells. The results we report here represent a milestone for designing artificial symbiosis of functionally active objects (such as micro-machines) in living cells. Moreover, our technique can be used for drug delivery, tissue engineering, and cell manipulation.     

Molecular identification of the causative agent of human strongyloidiasis acquired in Tanzania: Dispersal and diversity of Strongyloides spp. and their hosts

In order to identify the causative agent of imported strongyloidiasis found in a Japanese mammalogist, who participated in a field survey in Tanzania, the hyper-variable region IV (HVR-IV) of 18S ribosomal DNA and partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox1) were analyzed and compared with Strongyloides fuelleborni collected from apes and monkeys of Africa and Japan, and S. stercoralis from humans, apes and dogs. The HVR-IV and cox1 of the patient's worms were identical to or only slightly differed from those of worms parasitic in Tanzanian chimpanzees and yellow baboons, demonstrating that the patient acquired the infection during her field survey in Tanzania. Phylogenetic analysis with the maximum-likelihood method largely divided isolates of S. fuelleborni into three groups, which corresponded to geographical localities but not to host species. Meanwhile, isolates of S. stercoralis were grouped by the phylogenetic analysis into dog-parasitic and primate-parasitic clades, and not to geographical regions. It is surmised that subspeciation has occurred in S. fuelleborni during the dispersal of primates in Africa and Asia, while worldwide dispersal of S. stercoralis seems to have occurred more recently by migration and the activities of modern humans.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

SSA/Ro52 autoantigen interacts with Dcp2 to enhance its decapping activity

We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.     

Difference in aftereffects following prolonged Achilles tendon vibration on muscle activity during maximal voluntary contraction among plantar flexor synergists.

It has been suggested that a suppression of maximal voluntary contraction (MVC) induced by prolonged vibration is due to an attenuation of Ia afferent activity. The purpose of the present study was to test the hypothesis that aftereffects following prolonged vibration on muscle activity during MVC differ among plantar flexor synergists owing to a supposed difference in muscle fiber composition. The plantar flexion MVC torque and surface electromyogram (EMG) of the medial head of gastrocnemius (MG), the lateral head of gastrocnemius (LG), and the soleus (Sol) were recorded in 13 subjects before and after prolonged vibration applied to the Achilles tendon at 100 Hz for 30 min. The maximal H reflexes and M waves were also determined from the three muscles, and the ratio between H reflexes and M waves (H/Mmax) was calculated before and after the vibration. The MVC torque was decreased by 16.6 +/- 3.7% after the vibration (P < 0.05; ANOVA). The H/Mmax also decreased for all three muscles, indicating that Ia afferent activity was successfully attenuated by the vibration in all plantar flexors. However, a reduction of EMG during MVC was observed only in MG (12.7 +/- 4.0%) and LG (11.4 +/- 3.9%) (P < 0.05; ANOVA), not in Sol (3.4 +/- 3.0%). These results demonstrated that prolonged vibration-induced MVC suppression was attributable mainly to the reduction of muscle activity in MG and LG, both of which have a larger proportion of fast-twitch muscle fibers than Sol. This finding suggests that Ia-afferent activity that reinforces the recruitment of high-threshold motor units is necessary to enhance force exertion during MVC.     

Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance

Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this work, we characterized the organic solvent tolerance of Escherichia coli cells that overexpress prefoldin and group II chaperonin from a hyperthermophilic archeaum, Pyrococcus horikoshii OT3. The colony-forming efficiency of E. coli cells overexpressing prefoldin increased by 1,000-fold and decreased the accumulation of intracellular organic solvent. The effect was impaired by deletions of the region responsible for the chaperone function of prefoldin. Therefore, we concluded that prefoldin endows E. coli cells by preventing accumulation of intracellular organic solvent through its molecular chaperone activity.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Endogenous infection in mice with streptozotocin-induced diabetes. A feature of bacterial translocation

Slc:ddY mice that received a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ) were examined for persistency of diabetes (changes of indigenous bacterial floras, and bacterial translocation. Significant diabetes (increase in plasma glucose and decrease in insulin) was recognized 2 weeks after the injection, and persisted for 12 weeks. The numbers of aerobic gram-negative bacilli, staphylococci (including micrococci), and streptococci in caecal and oral floras were significantly increased, but the numbers of anaerobic bacteria in caecal flora were hardly changed. Bacterial translocation of indigenous bacteria to the mesenteric lymph node, lung, or kidney was detectable in some mice 2 weeks after the injection. The incidence of bacterial translocation in these STZ-treated mice then increased; infection caused by several organisms, e.g., Klebsiella pneumoniae, Staphylococcus epidermidis, streptococci, or Lactobacillus sp., occurred in lung, liver, spleen, kidneys, and mesenteric lymph node. No indigenous bacteria were cultured from these organs of control mice. This endogenous infection may have been due to the over population of several bacterial strains caused by disruption of indigenous floras along with depression of immunological function.