In Vitro Evaluation of the Effect of Stimulation with Magnetic Fields on Chondrocytes

Magnetic fields (MFs) have been used as an external stimulus to increase cell proliferation in chondrocytes and extracellular matrix (ECM) synthesis of articular cartilage. However, previously published studies have not shown that MFs are homogeneous through cell culture systems. In addition, variables such as stimulation times and MF intensities have not been standardized to obtain the best cellular proliferative rate or an increase in molecular synthesis of ECM. In this work, a stimulation device, which produces homogeneous MFs to stimulate cell culture surfaces was designed and manufactured using a computational model. Furthermore, an in vitro culture of primary rat chondrocytes was established and stimulated with two MF schemes to measure both proliferation and ECM synthesis. The best proliferation rate was obtained with an MF of 2 mT applied for 3 h, every 6 h for 8 days. In addition, the increase in the synthesis of glycosaminoglycans was statistically significant when cells were stimulated with an MF of 2 mT applied for 5 h, every 6 h for 8 days. These findings suggest that a stimulation with MFs is a promising tool that could be used to improve in vitro treatments such as autologous chondrocyte implantation, either to increase cell proliferation or stimulate molecular synthesis. Bioelectromagnetics. 2020;41:41–51 © 2019 Bioelectromagnetics Society     

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.     

Thermal stability of oligodeoxynucleotide duplexes containing l-deoxynucleotide at termini

The effects of substituting l-deoxynucleotide for d-deoxynucleotide at duplex termini were evaluated and the terminal substitutions were found to show much less effects on duplex destabilization and to show a similar tendency in base pairing selectivity, compared with internal chiral substitutions.     

Bio-functional pickles that reduce blood pressure of rats

Addition of γ-aminobutyric acid (GABA) and angiotensin converting enzyme (ACE)-inhibitory peptides to the pickles was studied in order to develop a new type of pickles that reduce blood pressure. Based on the outcome of these studies, a new type of fermentation bed composed of rice bran and white miso has been successfully developed. The advantage of such pickles is that they not only contain both GABA and ACE-inhibitory peptides, but also that their taste and flavor are excellent, with colors close to the original ones. The new type of pickles could temporarily reduce blood pressure in two types of rats, spontaneously hypertensive rats and NaCl-sensitive model rats. Thus, the newly developed pickles appear to be beneficial for pickle business.     

TGF-beta mediated Msx2 expression controls occipital somites-derived caudal region of skull development

Craniofacial development involves cranial neural crest (CNC) and mesoderm-derived cells. TGF-beta signaling plays a critical role in instructing CNC cells to form the craniofacial skeleton. However, it is not known how TGF-beta signaling regulates the fate of mesoderm-derived cells during craniofacial development. In this study, we show that occipital somites contribute to the caudal region of mammalian skull development. Conditional inactivation of Tgfbr2 in mesoderm-derived cells results in defects of the supraoccipital bone with meningoencephalocele and discontinuity of the neural arch of the C1 vertebra. At the cellular level, loss of TGF-beta signaling causes decreased chondrocyte proliferation and premature differentiation of cartilage to bone. Expression of Msx2, a critical factor in the formation of the dorsoventral axis, is diminished in the Tgfbr2 mutant. Significantly, overexpression of Msx2 in Myf5-Cre;Tgfbr2flox/flox mice partially rescues supraoccipital bone development. These results suggest that the TGF-beta/Msx2 signaling cascade is critical for development of the caudal region of the skull.     

Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.