Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives.

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an -hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe(OPh)]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-()-Phe(OPh) (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin.

Glioblastoma cell line shows phenotypes of cancer stem cells in hypoxic microenvironment of spheroids.

In this study, we examined the phenotypes of CD133-positive cells that were induced in a hypoxic microenvironment of spheroids formed using a glioblastoma cell line (T98G). Colony-formation assay showed that spheroid CD133-positive cells (SCPCs) were more resistant to X-rays and Temozolomide (TMZ) than spheroid CD133-negative cells (SCNCs) sorted from T98G spheroids. In contrast, the sensitivity to X-rays and TMZ was not different between hypoxic cells and normoxic cells of T98G spheroids in a colony-formation assay using green fluorescent protein (GFP) reporter-transfectants to monitor hypoxia.

Serum podocalyxin levels correlate with carotid intima media thickness, implicating its role as a novel biomarker for atherosclerosis.

Podocalyxin is a cell surface sialomucin, which is expressed in not only glomerular podocytes but also vascular endothelial cells. Urinary podocalyxin is used as a marker for glomerular disease. However, there are no reports describing serum podocalyxin (s-Podxl) levels.

The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium.

Glutathione (GSH) plays an important neuroprotective role, and its synthesis depends on the amount of available cysteine (CSH) in the cells. Various kinds of evidence suggest that astrocytes can provide CSH or GSH to neurons, but the delivery mechanism of the thiol-compounds has not been elucidated. In this study, the dynamics of CSH, GSH and their disulphides in astrocyte culture medium were investigated by following the time-course of concentration changes and by computer simulation and curve fitting to experimental data using a mathematical model.

Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells).

Introduction: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of (14)C(U)-L-tyrosine ((14)C-Tyr), (125)I-4-iodo-L-meta-tyrosine (4-(125)I-mTyr), (125)I-6-iodo-L-meta-tyrosine (6-(125)I-mTyr), (125)I-3-iodo-α-methyl-L-tyrosine ((125)I-IMT) and (125)I-3-iodo-L-tyrosine (3-(125)I-Tyr) using Chinese hamster ovary cells (CHO-K1).

Methods: Cells in the exponential growth phase were incubated with 18.

Conflicting effects of N-acetylcysteine on purified neurons derived from rat cortical culture.

We examined the protective effects of N-acetylcysteine (NAC) on the death of glia-free neurons in culture. Under normoxic conditions, the protection by NAC was observed only in cystine-free but not complete medium. When the cells were cultured under hypoxic conditions, NAC much elongated their survival even in the presence of cystine.

Stimulation of 125I-3-iodo-alpha-methyl-L-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters.

Introduction: Transport of the amino acid analog (123)I-3-iodo-alpha-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of (125)I-3-iodo-alpha-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells.

Methods: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.

Kinetics of hydrogen peroxide elimination by astrocytes and C6 glioma cells analysis based on a mathematical model.

Oxidative stress is implicated in a variety of disorders including neurodegenerative diseases, and H(2)O(2) is important in the generation of reactive oxygen and oxidative stress. In this study, we have examined the rate of extracellular H(2)O(2) elimination and relevant enzyme activities in cultured astrocytes and C6 glioma cells and have analyzed the results based on a mathematical model. As compared with other types of cultured cells, astrocytes showed higher activity of glutathione peroxidase (GPx) but lower activities for GSH recycling.

Transport of D-[1-14C]-amino acids into Chinese hamster ovary (CHO-K1) cells: implications for use of labeled d-amino acids as molecular imaging agents.

Introduction: The fact that d-amino acids have been found in various tissues and are involved in various functions is a clue to how to develop new imaging agents. We examined d-amino acid transport mechanisms in Chinese hamster ovary (CHO-K1) cells because CHO-K1 cells are widely used in biomedical studies and are thought to be useful for expression of genes involved in metabolism of D-amino acids.

Methods: Uptake experiments were performed.

Glutathione induces neuronal differentiation in rat bone marrow stromal cells.

It has been reported that rat bone marrow stromal cells (BMSCs) are differentiated into neuronal cells by administration of 2-mercaptoethanol [Woodbury et al (2000) J Neurosci Res 61:364-370]. In this study, we examined the effects of various sulfhydryl (SH) compounds on the differentiation of BMSCs obtained from rat femurs. Neuronal differentiation was detected morphologically and immunocytochemically.

Expression and function of cystine/glutamate transporter in neutrophils.

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS-mediated oxidant injury. Cystine/glutamate transporter, designated as system xc- and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian-cultured cells.

Beneficial effect of antioxidants in purified neurons derived from rat cortical culture.

Brain cell suspensions obtained from cerebrum of fetal rats were cultured and after 5 days neurons were separated from the residual cells. These purified neurons, which were replated on the dish, started to die within 24 h in culture. Glutathione content of these neurons decreased rapidly to less than one-tenth of the initial level after 24 h.