Cellular Culture Medium Research Articles

Improvement of monoclonal antibody stability by modulating trace metal iron concentration in cell culture media: A case study

Monoclonal antibodies are among the fastest-growing segment of drugs. The stability of the protein drugs is particularly challenging due to their high susceptibility to physico-chemical stresses that may make them unsuitable for human health applications. Aggregation is considered a main stability indicator, potentially raising safety and immunogenicity concerns. Self-aggregation pathways can be triggered by a series of protein modifications that could be due to the intrinsic characteristics of the protein itself or during the manufacturing process. Macro and micronutrient content in cellular culture media can represent a source of oxidative stress, leading to a higher self-aggregation rate. This work aimed to explore the impact of iron content variability in the manufacturing cell culture media on drug substance purity and quality, and linked the effect of iron content on antibody degradation by self-aggregation, methionine oxidation, and decreased stability under long-term conditions. This work highlights the importance of maintaining strict control of raw materials, as unexpected lot-to-lot variability could compromise the entire antibody manufacturing process and patient safety due to undesired issues in the quality of the final product.

Synthesis of Composite of Hydroxyapatite using Chitosan Template

This work presents the synthesis of composite HA in chitosan polymer. The synthetic composite was investigated by using XRD, FTIR, SEM methods. The experiment for bio-compatibility was effectuated by the contacting of the extract solutions from synthetic composite and the cellular culture media. The obtained results confirmed the introduction of both HA and Chitosan in the structure of synthetic composite. The high values of cellular viability confirmed the good bio-compatibility of synthetic composite.

РІВЕНЬ ГЛЮКОЗИ ТА ГАММА-ГЛУТАМІЛТРАНСПЕПТИДАЗНА АКТИВНІСТЬ У КЛІТИНАХ ГЕПАТОЦИТАРНОГО ПОХОДЖЕННЯ ЗА ДІЇ ЕКСТРАКТІВ ТА ЦИТОКІНІНОВИХ ФРАКЦІЙ ЛІКАРСЬКИХ ГРИБІВ

Mushroom extracts show the multifunctional activity and have a wide range of applications for the treatment of various diseases, including cancer. However, the full composition of the compounds that produce macromycetes that exhibit antitumor properties has not yet been established. Impaired glucose metabolism and activation of gamma-glutamyltranspeptidase (GGT) in tumor cells may be a key marker of biochemical anaplasia in neoplasms. The aim of the study was to investigate the effect of crude extracts and cytokinin fractions isolated from the mycelial biomass of medicinal mushrooms on the biological properties of cells of hepatocyte-like cells of the HepG2 line (human hepatocellular carcinoma). The objects of the research were pure mushroom cultures of Hericium coralloides, Fomitopsis officinalis, Trametes (Coriolus) versicolor, Pleurotus ostreatus and Morchella esculenta. Cytokinin fractions from the extracts were isolated by centrifugation followed by fractionation and purification using ion exchange chromatography. Qualitative and quantitative analysis of cytokinins was performed by high-performance liquid chromatography. GGT activity was determined using the kit "Filisit" (Ukraine), glucose level – glucose oxidase method, with modifications for the cellular culture medium. The analysis of mycelial biomass of medicinal macromycetes revealed the presence of transzeatin, zeatin riboside, zeatin-O-glucoside and isopentenyladenine, that showed high activity in relation to cytokinin synthesis. Inhibition of glucose diffusion from the cultivation medium with the use of crude extracts and cytokinin fractions of medicinal mushroom and a decrease in GGT activity, more pronounced with the action of cytokinin fractions, compared with crude extracts, was noted. The difference between the effects of crude extracts and cytokinin fractions indicated the complex nature of the action of biologically active substances of medicinal mushroom. Presented results regarding the effects of crude extracts and cytokinin fractions of medicinal mushroom showed a normalizing effect on the main metabolic parameters which change in tumor cells, as a mechanism of biochemical anaplasia.

Electrospun conductive gold covered polycaprolactone fibers as electrochemical sensors for O2 monitoring in cell culture media

This work reports the use of electrospun conductive gold covered polycaprolactone fibers for the quantification of dissolved O2. The morphologies of the electrospun fibers obtained at a static and a dynamic drum collector were investigated by scanning electron microscopy. The reduction process of O2 at negative potentials is analyzed by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) in sodium phosphate buffer (NaPB) pH 7.0 and in cellular media pH 7.4. The electrochemical sensing performance of Au/PCL towards O2 quantification in NaPB and cellular media is compared by using three electrochemical techniques: cyclic and linear sweep voltammetry and EIS. Measurements are done in a two electrode configuration, using a silver wire as reference, to show the applicability of the method for O2 quantification in cellular culture media.

Organic Electrochemical Transistor for in Situ Detection of H2O2 Released from Adherent Cells and Its Application in Evaluating the In Vitro Cytotoxicity of Nanomaterial

Detection of hydrogen peroxide (H2O2) produced by living cells is very significant to fully understand its roles in cellular physiology, as well as providing reliable diagnosis of pathological conditions. However, in situ detection of H2O2 released from adherent cells in cellular culture medium is still insufficiently achieved. Here, we report an electrochemical platform for in situ detection of H2O2 produced by adherent cells in cellular culture medium. It is based on the use of organic electrochemical transistor (OECT) fabricated on a flexible poly(ethylene terephthalate) substrate and Transwell support. A screen-printed carbon paste electrode was modified with carbon nanotubes and platinum nanoparticles and served as the gate of the device. Under optimal conditions, this device exhibits good modulation and sensitivity. It works in the 0.5 μM to 0.1 mM H2O2 concentration range and has a 0.2 μM detection limit. The cells were seeded and grew on the Transwell membrane. Upon being stimulated by N-formylmethionyl-leucyl-phenylalanine peptide, H2O2 produced by the adherent cells diffused into the bottom chamber of the Transwell and was in situ detected by OECT. Moreover, evaluating in vitro cytotoxicity of the nanomaterial using the OECT-Transwell platform was realized. This simple electrochemical platform would be of great interest for in vitro cytotoxicity, cellular physiology study, and diagnosis of pathological conditions.

Improving dispersive property, biocompatibility and targeting gene transfection of graphene oxide by covalent attachment of polyamidoamine dendrimer and glycyrrhetinic acid

The surface functional groups of GO have significant effects on the performances of GO-based gene delivery vector. In this work, the polyamidoamine (PAMAM) dendrimer and glycyrrhetinic acid (GA) were tethered onto the GO surface by one-step covalently cross-linking method. The micro-morphology, surface functional groups, and zeta potential of the obtained GO-PAMAM-GA hybrid were characterized and verified. The effects of GA amount in the hybrid on the dispersive property in cell culture medium, in vitro cytotoxicity to human hepatocarcinoma (SMMC-7721) and human embryonic kidney (HEK-293) cells, and gene (plasmid DNA of enhanced green fluorescent protein) transfection capacity were investigated in detail. Under optimal conditions, the obtained hybrid shows small average size (about 160 nm) and has very good dispersive stability (in 30 days) in cellular culture medium. Compared with the GO-PAMAM without GA modification, the GO-PAMAM-GA hybrid exhibits greatly enhanced biocompatibility to the two cell lines. The cellular viability of SMMC-7721 cells still retains about 98% even the concentration of the hybrid up to 200 μg mL-1. The gene transfection capacity of the GO-PAMAM has been improved about 50% through the GA functionalization. Moreover, the GO-PAMAM-GA hybrid possesses targeting gene transfection to SMMC-7721 cells.

Overexpression of miR-21 in stem cells improves ovarian structure and function in rats with chemotherapy-induced ovarian damage by targeting PDCD4 and PTEN to inhibit granulosa cell apoptosis

BackgroundChemotherapy-induced premature ovarian failure (POF) is a severe complication affecting tumor patients at a childbearing age. Mesenchymal stem cells (MSCs) can partially restore the ovarian structure and function damaged by chemotherapy. miR-21 is a microRNA that can regulate cell apoptosis. This study discusses the repair effect and mechanism of MSCs overexpressing miR-21 on chemotherapy-induced POF.MethodsRat MSCs and granulosa cells (GCs) were isolated in vitro. MSCs were transfected with miR-21 lentiviral vector (LV-miR-21) to obtain MSCs stably expressing miR-21 (miR-21-MSCs). The microenvironment of an ovary receiving chemotherapy was mimicked by adding phosphamide mustard (PM) into the cellular culture medium. The apoptosis rate and the mRNA and protein expression of target genes PTEN and PDCD4 were detected in MSCs. Apoptosis was induced by adding PM into the culture medium for GCs, which were cocultured with miR-21-MSCs. The apoptosis rate and the mRNA and protein expression of PTEN and PDCD4 were detected. The chemotherapy-induced POF model was built into rats by intraperitoneal cyclophosphamide injection. miR-21-MSCs were transplanted into the bilateral ovary. The rats were sacrificed at 15, 30, 45, and 60 days after the last injection. The ovarian weights, follicle count, estrous cycle, and sex hormone levels (estradiol (E2) and follicle-stimulating hormone (FSH)) were detected. Apoptosis of GCs was determined by TUNEL assay. The miR-21 and mRNA and protein expression of PTEN and PDCD4 were determined.ResultsThe apoptosis decreased in MSCs transfected with miR-21. The mRNA and protein expression of target genes PTEN and PDCD4 was downregulated. GCs cocultured with miR-21-MSCs showed a decreased apoptosis, an upregulation of miR-21, and a downregulation of PTEN and PDCD4. Following the injection of miR-21-MSCs, the ovarian weight and follicle counts increased; E2 levels increased while FSH levels decreased, with less severe apoptosis of GCs. The miR-21 expression in the ovaries was upregulated, while the mRNA expression and protein expression of PTEN and PDCD4 were downregulated.ConclusionsOverexpression of miR-21 in MSCs promoted efficacy against chemotherapy-induced POF and its improvement of the repair effect was related to the inhibition of GC apoptosis by targeting PTEN and PDCD4.

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

PPAR-α agonist regulates amyloid-β generation via inhibiting BACE-1 activity in human neuroblastoma SH-SY5Y cells transfected with APPswe gene.

Alzheimer's disease is a neuroinflammatory disease and is the most common cause of dementia in the elderly. Studies have shown the beneficial effects of the peroxisome proliferator-activated receptor alpha (PPAR-α) agonists on the treatment of neuroinflammatory diseases. The aim of the present study is to examine the ability of GW7647 (a PPAR-α agonist) to regulate amyloid precursor protein (APP) amyloidogenic processing in human neuroblastoma SH-SY5Y cells transfected with APPswe gene. After administration of GW7647 for 24h, the levels of APP, soluble APPβ (sAPPβ), and presenilin 1 (PS-1) were assessed by Western blot. Cellular culture medium levels of amyloid-β 42 (Aβ42) were analyzed by ELISA, and the activity of beta-site APP cleaving enzyme 1 (BACE-1) was measured by fluorometric assay. We found that GW7647 decreased the expression of sAPPβ and the activity of BACE-1, and also reduced Aβ42 release. However, GW7647 did not modify the levels of APP and PS-1. Furthermore, LY294002, the phosphoinositide 3-kinase (PI3-K) inhibitor, reversed the effects of GW7647 on the BACE-1 activity and the levels of sAPPβ and Aβ42. Our data demonstrate that GW7647 may reduce Aβ production via inhibiting BACE-1 activity, and this may involve in PI3-K pathway.

A pH-responsive layered double hydroxide (LDH)-phthalocyanine nanohybrid for efficient photodynamic therapy.

A pH-responsive nanohybrid (LDH-ZnPcPS4 ), in which a highly hydrophilic zinc(II) phthalocyanine tetra-α-substituted with 4-sulfonatophenoxy groups (ZnPcPS4 ) is incorporated with a cationic layered double hydroxide (LDH) based on electrostatic interaction, has been specially designed and prepared through a facile co-precipitation approach. ZnPcPS4 is an excellent singlet-oxygen generator with strong absorption at the near-infrared region (692 nm) in cellular culture media, whereas the photoactivities of ZnPcPS4 were remarkably inhibited after incorporation with the LDH. The nanohybrid is essentially stable in aqueous media at pH 7.4; nevertheless, in slightly acidic media of pH 6.5 or 5.0, ZnPcPS4 can be efficiently released from the LDH matrix, thus leading to restoration of the photoactivities. The nanohybrid shows a high photocytotoxicity against HepG2 cells as a result of much more efficient cellular uptake and preferential accumulation in lysosomes, whereby the acidic environment leads to the release of ZnPcPS4 . The IC50 value of LDH-ZnPcPS4 is as low as 0.053 μM, which is 24-fold lower than that of ZnPcPS4 . This work provides a facile approach for the fabrication of photosensitizers with high photocytotoxicity, potential tumor selectivity, and rapid clearance character.

Extraction of alpha-fetoprotein (AFP) with magnetic albuminoid-imprinted poly(ethylene-co-vinyl alcohol) nanoparticles from human hepatocellular carcinoma HepG2 cellular culture medium

The selective adsorption of alpha-fetoprotein using albuminoid MMIP nanoparticles from the human hepatocellular carcinoma HepG2 cellular culture medium.

Osteodifferentiation of mesenchymal stem cells on chitosan/hydroxyapatite composite films

Chitosan (Ch) is one of the most commonly used natural biomaterials. Osteodifferentiation of mesenchymal stem cells (MSCs) on Ch has drawn extensive interest. Hydroxyapatite (HA) is a component of skeleton and teeth with good biocompatibility. Combination with HA may be a good method to modify Ch to facilitate cellular behaviors and functions on it. In this study, Ch/HA film was prepared and characterized. Its potential to benefit cellular behaviors and osteodifferentiation of MSCs was evaluated. Resultantly, physical properties of composite Ch/HA, including water-in-air contact angle, tensile strength, elastic modulus, and breaking elongation were favorably modified. In cellular culture medium, Ch/HA films absorbed more Ca(2+) than Ch films, and more HA crystalline growths on Ch/HA films. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and morphological features showed better proliferation and adhesion of MSCs on Ch/HA films. Osteodifferentiation of MSCs on Ch/HA was promoted, indicated by modified transcription level of osteocalcin, osteopontin, collagen I, alkaline phosphatase (ALP), and induced ALP activity. These data suggest biocompatibility of Ch is modified after being blended with HA, which promotes osteodifferentiation of MSCs. This can be a promising approach to modify Ch for its applications in bone tissue engineering.

Cell viability studies and operation in cellular culture medium of n-type organic field-effect transistors

The possibility of the fabrication of organic devices suitable to be applied in bio-sensing fields depends largely on the availability of organic compounds displaying robust electrical properties even in aqueous solutions and effective biocompatibility features. In this paper, we report about the good cellular biocompatibility and the electrical response stability in an ionic medium of n-type organic transistors based on the recently developed PDI-8CN2 oligomer. The biocompatibility has been tested by analyzing the adhesion and viability of two different cell lines, human epithelial HeLa cells and murine neuronal F11 cells, on PDI-8CN2 films grown by organic molecular beam deposition (OMBD) on SiO2 substrates. The effect of film thickness on cell attachment was also tested. Uncoated SiO2 substrates were used as control surfaces and sexithiophene (T6) as device testing control. Moreover, the possible toxicity of –CN groups of PDI-8CN2 was tested on HeLa cell cultures, using PDI-8 and T6 molecules as controls. Results showed that, although at high concentration these organic compounds are toxic in solution, if they are presented in form of film, cell lines can attach and grow on them. The electrical response stability of PDI-8CN2 transistors in a cellular culture medium characterized by high concentrations of ionic species has been also investigated. For this purpose, low-voltage operation devices with VGS ranging from −5 V to 5 V, able to strongly reduce the influence of Faradaic currents coming from the electrical operation in an highly ionic environment, have been fabricated on 35 nm thick SiO2 layers and electrically characterized. These results are useful to experimentally define the main critical issues to be further addressed for the fabrication of reliable bio-sensors based on organic transistors.

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)(3)](2+), was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)(3)](2+) characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Hydrogen sulfide reduces mRNA and protein levels of beta-site amyloid precursor protein cleaving enzyme 1 in PC12 cells

Hydrogen sulfide (H 2S) is now identified as a new neuromodulator. Increasing evidence suggest that H 2S may play an important role in the progression of Alzheimer's disease (AD). The aim of the present study is to investigate the effects of H 2S on beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) expression and amyloid beta (Aβ) secretion in PC12 cells. The levels of BACE-1 mRNA were measured by quantitative polymerase chain reaction analysis. BACE-1 protein levels were assessed by Western blot. Cellular culture medium levels of Aβ1-42 were analyzed by ELISA. We found that sodium hydrosulfide (NaHS), a H 2S donor, decreased BACE-1 mRNA and protein levels and Aβ1-42 release. Furthermore, NaHS promoted the phosphorylation of Akt and ERK but not JNK or p38 MAPK. However, the effects of NaHS on BACE-1 expression and Aβ1-42 secretion were abolished by inhibitors of phosphatidylinositol 3-kinase (PI3-K), but not of mitogen-activated protein kinase kinases (MEK). Our data indicate that H 2S reduces BACE-1 expression in PC12 cells via activation of PI3-K/Akt signaling pathways. H 2S releasing drugs may have therapeutic potential in AD patients.

Enhanced Cytotoxicity of Core Modified Chitosan Based Polymeric Micelles for Doxorubicin Delivery

In this study, the cytotoxicity of doxorubicin (DOX) loaded stearic acid grafted chitosan oligosaccharide (CSO‐SA) micelles and its core modified drug delivery systems were investigated in vitro. The in vitro drug release experiments using cellular culture medium, Roswell Park Memorial Institute 1640 (RPMI‐1640) medium as a dissolution medium confirmed that the DOX release from CSO‐SA micelles was successfully delayed by the core modification of CSO‐SA micelles with stearic acid (SA). The cell viability assay against A549 cells indicated the 50% inhibition concentration (IC50) of blank CSO‐SA micelles and the core modified CSO‐SA micelles was 369 ± 27 µg/mL and 234 ± 9 µg/mL, respectively. The entrapment of DOX by CSO‐SA micelles could decrease the IC50 of DOX from 3.5 to 1.9 µg/mL, and a further reduction to 0.7 µg/mL could result by the core modification of CSO‐SA micelles. The fluorescence image observations of DOX and DOX concentration measurements inside A549 cells demonstrated that the DOX concentration inside cells was increased by the entrapment of CSO‐SA micelles, and further enhanced by the core modification of CSO‐SA micelles. The results indicated that the CSO‐SA micelles with modified cores could be useful as a drug delivery vehicle for cancer chemotherapy. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:704–712, 2009

Comparison between non-peripherally and peripherally tetra-substituted zinc (II) phthalocyanines as photosensitizers: Synthesis, spectroscopic, photochemical and photobiological properties

A series of zinc phthalocyanines tetra-α-substituted with 4-(butoxycarbonyl) phenoxy groups ( 1a) or 4-carboxylphenoxy groups ( 2a) or 4-(2-carboxyl-ethyl)phenoxy groups ( 3a), and the corresponding tetra-β-substituted ( 1– 3b) analogues, have been synthesized and characterized. The effects of the position of substituents at the phthalocyanine skeleton on their spectroscopic, photochemical and photobiological properties have been revealed. When compared with the tetra-β-substituted phthalocyanines, the corresponding tetra-α-substituted analogues exhibit a less aggregating trend in the cellular growth medium, a slightly higher singlet oxygen quantum yield and higher photo-stability in DMF, and a comparable cellular uptake. As a result, the tetra-α-substituted zinc phthalocyanines exhibit a higher photocytotoxicity toward MGC803 human gastric carcinoma cells than the tetra-β-substituted counterparts. Among all these compounds, phthalocyanine 2a shows the highest photodynamic activity, which may mainly be due to its non-aggregated nature in cellular culture medium and high cellular uptake.

Enhancement of the thermal transport in a culture medium with Au nanoparticles

In this work, it is reported the gold nanoparticles synthesis, their characterization, and their application to the enhancement of the thermal transport in a cellular culture medium. The Au nanoparticles (NPs), with average size of 10 nm, contained into a culture medium (DMEM (1)/F12(1)) (CM) increased considerably the heat transfer in the medium. Thermal lens spectrometry (TLS) was used to measure the thermal diffusivity of the nanofluids. The characteristic time constant of the transient thermal lens was obtained by fitting the theoretical expression, for transient thermal lens, to the experimental data. Our results show that the thermal diffusivity of the culture medium is highly sensitive to the Au nanoparticle concentration and size. The ability to modify the thermal properties to nanometer scale becomes very important in medical applications as in the case of cancer treatment by using photodynamic therapy (PDT). A complementary study with UV–vis and TEM techniques was performed to characterize the Au nanoparticles.

Novel Processing and Localization of catA, ccdA Associated Thiol-Disulfide Oxidoreductase, in Protein Hyper-Producing Bacterium Brevibacillus choshinensis

Previously, we have cloned ccdA and its associated thiol-disulfide oxidoreductase gene, catA, in Brevibacillus choshinensis. CcdA is known to be an integral membrane protein and its associated oxidoreductase homologues are believed to be membrane anchoring proteins, both providing reducing equivalents across the membrane to control correct disulfide bond formation. Here, we found that CatA is first localized as a membrane bound form and then slowly released into the cellular periphery and culture medium with cleavage at a novel processing site.