Cyclodextrin-responsive nanogel as an artificial chaperone for horseradish peroxidase

The thermal stabilization and refolding of horseradish peroxidase (HRP) upon heating were investigated using an artificial molecular chaperone consisting of cholesterol-bearing pullulan (CHP) nanogels. The CHP nanogels inhibited the aggregation of HRP under heating by complexation with the denatured HRP. The enzyme activity of HRP complexed with CHP nanogels was not detected. However, the enzyme activity recovered up to 80% of native HRP after the addition of cyclodextrin (CD) to the complex. The dissociation of CHP nanogels was induced by the formation of an inclusion complex of cholesterol groups of CHP with CD. The enzyme activity of HRP was only significantly recovered by the addition of β-CD or its derivatives. Natural molecular chaperones, such as GroEL/ES, trap, fold, and release the nonnative proteins by changing the hydrophobicity of the specific sites of the molecular chaperone that interact with the nonnative protein. The functional mechanism of the nanogel chaperon system is similar to that of natural molecular chaperones. The nanogel chaperone system is a useful tool to aid the refolding and thermal stabilization of unstable proteins for post-genome research, and in medical and biological applications

Expression of tyrosine hydroxylase in cerebellar Purkinje cells of ataxic mutant mice:its relation to the onset and/or development of ataxia

This report describes recent studies on tyrosine hydroxylase (TH) expression in Purkinje cells of the cerebellum of ataxic mutant mice. An increased expression of TH in some Purkinje cells has been observed in two allelic groups of mutant mice, tottering and dilute. TH-positive Purkinje cells appeared preceding the onset of ataxia. Northern blot analysis revealed2.1kb of TH mRNA in the mutant cerebella, and the size was identical to that of TH transcripts in other brain regions. However, TH in Purkinje cells did not seem to participate in catecholamine biosynthesis. In vitro studies showed that cultured non-catecholaminergic neurons expressed the TH transcripts following Ca2+ influx. Therefore, abnormal TH expression in the mutant Purkinje cells may indicate neuronal dysfunction caused by misregulation of intracellular Ca2+concentrations

Development and single‐particle analysis of hybrid extracellular vesicles fused with liposomes using viral fusogenic proteins

Extracellular vesicles (EVs) have potential biomedical applications, particularly as a means of transport for therapeutic agents. There is a need for rapid and efficient EV-liposome membrane fusion that maintains the integrity of hybrid EVs. We recently described Sf9 insect cell-derived EVs on which functional membrane proteins were presented using a baculovirus-expression system. Here, we developed hybrid EVs by membrane fusion of small liposomes and EVs equipped with baculoviral fusogenic proteins. Single-particle analysis of EV-liposome complexes revealed controlled introduction of liposome components into EVs. Our findings and methodology will support further applications of EV engineering in biomedicine

Reversible conjugation of biomembrane vesicles with magnetic nanoparticles using a self-assembled nanogel interface: single particle analysis using imaging flow cytometry

Nanoscale biomembrane vesicles such as liposomes and extracellular vesicles are promising materials for therapeutic delivery applications. However, modification processes that disrupt the biomembrane affect the performance of these systems. Non-covalent functionalization approaches that are facile and easily reversed by environmental triggers are therefore being widely investigated. In this study, liposomes were successfully hybridized with magnetic iron oxide particles using a cholesterol-modified pullulan nanogel interface. Both the magnetic nanoparticles and the hydrophobic core of the lipid bilayer interacted with the hydrophobic cholesteryl moieties, resulting in stable hybrids after simple mixing. Single particle analysis by imaging flow cytometry showed that the hybrid particles interacted in solution. Calcein loaded liposomes were not disrupted by the hybridization, showing that conjugation did not affect membrane stability. The hybrids could be magnetically separated and showed significantly enhanced uptake by HeLa cells when a magnetic field was applied. Differential scanning calorimetry revealed that the hybridization mechanism involved hydrophobic cholesteryl inserting into the biomembrane. Furthermore, exposure of the hybrids to fetal bovine serum proteins reversed the hybridization in a concentration dependent manner, indicating that the interaction was both reversible and controllable. This is the first example of reversible inorganic material conjugation with a biomembrane that has been confirmed by single particle analysis. Both the magnetic nanogel/liposome hybrids and the imaging flow cytometry analysis method have the potential to significantly contribute to therapeutic delivery and nanomaterial development

Change in Long-Spacing Collagen in Descemet's Membrane of Diabetic Goto-Kakizaki Rats and Its Suppression by Antidiabetic Agents

We examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen (type I, III, IV, VI, and VIII), laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in the density of the long-spacing collagen. Immunoelectron microscopy showed that type VIII collagen was localized in the internodal region of the long-spacing collagen, which was not labelled by any of the other antibodies used. The antidiabetic agents nateglinide and glibenclamide significantly suppressed the formation of the long-spacing collagen in the diabetic rats. Long-spacing collagen would thus be a useful indicator for studying diabetic changes in the cornea and the effect of antidiabetic agents

Increases in Nonspecific Immunoglobulin E and Eosinophils after H. pylori Eradication

Helicobacter pylori infection has been reported to be inversely associated with allergic disorders. We by chance experienced a patient with atrophic gastritis who presented marked elevations of both nonspecific serum immunoglobulin E and eosinophil counts after H. pylori eradication. A 49-year-old Japanese man received eradication of H. pylori using lansoprazole 60 mg/day, amoxicillin 1,500 mg/day, and clarithromycin 400 mg/day for 7 days. Serum immunoglobulin E increased to more than four times its pretreatment level, 306 → 485 → 1,325 U/ml, and peripheral eosinophil counts increased to more than three times, 99 → 139 → 298 per μl. Deducing from the current case, H. pylori eradication might develop allergic disorders in some patients

Ca2＋チャンネルビョウ マウス ニオケル ショウノウ ノ イジョウ ト ウンドウ シッチョウ

This review summarizes recent studies on the morphological abnormalities of cerebella in four ataxic mutant mice, i.e., tottering mouse, leaner mouse, rolling mouse Nagoya (RMN) and rocker mouse. These mutants carry mutations in the Ca2+ channel α1A subunit gene, and become useful models for human Ca2+ channelopathy such as episodic ataxia type-2 and familial hemiplegic migraine. Abnormal expression of tyrosine hydroxylase (TH) in some Purkinje cells has been observed in tottering mice, leaner mice and RMN, but not in rocker mice. However, Purkinje cells did not seem to synthesize catecholamines. Since the transcription of the TH gene is facilitated by Ca2+, TH expression in the mutant Purkinje cells indicates functional abnormality by alterations in intracellular Ca2+ concentrations. Corticotropin-releasing factor (CRF) immunoreactivity in some climbing or mossy fibers was higher in RMN than in controls. Double immunostaining for CRF and TH revealed a correspondence in the distribution of TH-positive Purkinje cells to terminal fields of CRF-positive climbing fibers in RMN. Therefore, CRF seems to alter granule and Purkinje cell functions, such as abnormal TH expression, indicating the possible expression of ataxic symptoms

Cold-induced metabolic conversion of haptophyte di- to tri-unsaturated C37 alkenones used as palaeothermometer molecules

The cosmopolitan marine haptophyte alga Emiliania huxleyi accumulates very long-chain (C37-C40) alkyl ketones with two to four trans-type carbon-carbon double bonds (alkenones). These compounds are used as biomarkers of haptophytes and as palaeothermometers for estimating sea-surface temperatures in biogeochemistry. However, the biosynthetic pathway of alkenones in algal cells remains enigmatic, although it is well known that the C37 tri-unsaturated alkenone (K37:3) becomes dominant at low temperatures, either by desaturation of K37:2 or by a separate pathway involving the elongation of tri-unsaturated alkenone precursors. Here, we present experimental evidence regarding K37:3 synthesis. Using the well-known cosmopolitan alkenone producer E. huxleyi, we labelled K37:2 with 13C by incubating cells with 13C-bicarbonate in the light at 25 °C under conditions of little if any K37:3 production. After stabilisation of the 13C-K37:2 level by depleting 13C-bicarbonate from the medium, the temperature was suddenly reduced to 15 °C. The 13C-K37:2 level rapidly decreased, and the 13C-K37:3 level increased, whereas the total 13C-K37 level—namely [K37:2 + K37:3]—remained constant. These 13C-pulse-chase-like experimental results indicate that 13C-K37:2 is converted directly to 13C-K37:3 by a desaturation reaction that is promoted by a cold signal. This clear-cut experimental evidence is indicative of the existence of a cold-signal-triggered desaturation reaction in alkenone biosynthesis