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.     

Basic pH-induced modification of excitation-energy dynamics in fucoxanthin chlorophyll a/c-binding proteins isolated from a pinguiophyte,Glossomastix chrysoplasta

Photosynthetic organisms have diversified light-harvesting complexes (LHCs) to collect solar energy efficiently, leading to an acquisition of their ecological niches. Herein we report on biochemical and spectroscopic characterizations of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a marine pinguiophyte Glossomastix chrysoplasta. The pinguiophyte FCP showed one subunit band in SDS-PAGE and one protein-complex band with a molecular weight at around 66 kDa in clear-native PAGE. By HPLC analysis, the FCP possesses chlorophylls a and c, fucoxanthin, and violaxanthin. To clarify excitation-energy-relaxation processes in the FCP, we measured time-resolved fluorescence spectra at 77 K of the FCP adapted to pH 5.0, 6.5, and 8.0. Fluorescence curves measured at pH 5.0 and 8.0 showed shorter lifetime components compared with those at pH 6.5. The rapid decay components at pH 5.0 and 8.0 are unveiled by fluorescence decay-associated (FDA) spectra; fluorescence decays occur in the 270 and 160-ps FDA spectra only at pH 5.0 and 8.0, respectively. In addition, energy-transfer pathways with time constants of tens of picoseconds are altered under the basic pH condition but not the acidic pH condition. These findings provide novel insights into pH-dependent energy-transfer and energy-quenching machinery in not only FCP family but also photosynthetic LHCs.     

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD⁺-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD⁺ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD⁺-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP⁺ specificity, suggesting their key roles in coenzyme specificity.     

Internalization of isolated functional mitochondria: involvement of macropinocytosis

In eukaryotic cells, mitochondrial dysfunction is associated with a variety of human diseases. Delivery of exogenous functional mitochondria into damaged cells has been proposed as a mechanism of cell transplant and physiological repair for damaged tissue. We here demonstrated that isolated mitochondria can be transferred into homogeneic and xenogeneic cells by simple co‐incubation using genetically labelled mitochondria, and elucidated the mechanism and the effect of direct mitochondrial transfer. Intracellular localization of exogenous mitochondria was confirmed by PCR, real‐time PCR, live fluorescence imaging, three‐dimensional reconstruction imaging, continuous time‐lapse microscopic observation, flow cytometric analysis and immunoelectron microscopy. Isolated homogeneic mitochondria were transferred into human uterine endometrial gland‐derived mesenchymal cells in a dose‐dependent manner. Moreover, mitochondrial transfer rescued the mitochondrial respiratory function and improved the cellular viability in mitochondrial DNA‐depleted cells and these effects lasted several days. Finally, we discovered that mitochondrial internalization involves macropinocytosis. In conclusion, these data support direct transfer of exogenous mitochondria as a promising approach for the treatment of various diseases.     

Are Additional Trace Elements Necessary in Total Parenteral Nutrition for Patients with Esophageal Cancer Receiving Cisplatin-Based Chemotherapy?

It is known that cisplatin induces the excretion of zinc from the urine and thereby reduces its serum concentration. However, the fluctuation of these trace elements during or after cisplatin-based chemotherapy has not been evaluated. To answer this question, we performed a clinical study in esophageal cancer patients undergoing cisplatin-based chemotherapy. Eighteen patients with esophageal cancer who were not able to swallow food or water orally due to complete stenosis of the esophagus were evaluated. The patients were divided into a control group [total parenteral nutrition (TPN) alone for 28?days, ten cases] and an intervention group (TPN with additional trace elements for 28?days, eight cases). The serum concentrations of zinc, iron, copper, manganese, triiodothyronin (T3), and thyroxin (T4), as alternative indicators of iodine, were measured on days?0, 14, and 28 of treatment, and statistically analyzed on day?28. In the control group, the serum concentration of copper was significantly decreased from 135.4 (day?0) to 122.1???g/ml (day?14), and finally to 110.6???g/ml (day?28, p?=?0.015). The concentration of manganese was also significantly decreased from 1.34 (day?0) to 1.17???g/ml (day?14) and finally to 1.20 (day?28, p?=?0.049). The levels of zinc, iron, T3, and T4 were not significantly changed. In the intervention group, the supplementation with trace elements successfully prevented these decreases in their concentrations. TPN with supplementary trace elements is preferable and recommended for patients who are undergoing chemotherapy in order to maintain the patients?? nutrient homeostasis.     

A new method for measuring torsional deformity in scoliosis

Background

This study evaluates the outcome and complications of decompressive cervical Laminectomy and lateral mass screw fixation in 110 cases treated for variable cervical spine pathologies that included; degenerative disease, trauma, neoplasms, metabolic-inflammatory disorders and congenital anomalies.

Methods

A retrospective review of total 785 lateral mass screws were placed in patients ages 16-68 years (40 females and 70 males). All cases were performed with a polyaxial screw-rod construct and screws were placed by using Anderson-Sekhon trajectory. Most patients had 12-14-mm length and 3.5 mm diameter screws placed for subaxial and 28-30 for C1 lateral mass. Screw location was assessed by post operative plain x-ray and computed tomography can (CT), besides that; the facet joint, nerve root foramen and foramen transversarium violation were also appraised.

Results

No patients experienced neural or vascular injury as a result of screw position. Only one patient needed screw repositioning. Six patients experienced superficial wound infection. Fifteen patients had pain around the shoulder of C5 distribution that subsided over the time. No patients developed screw pullouts or symptomatic adjacent segment disease within the period of follow up.

Conclusion

decompressive cervical spine laminectomy and Lateral mass screw stabilization is a technique that can be used for a variety of cervical spine pathologies with safety and efficiency.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.