Uptake of dehydroascorbic acid in high-GSH and normal-GSH dog erythrocytes

Uptake of dehydroascorbic acid (DHA) was studied in two types of dog erythrocytes with high GSH and normal GSH levels. Compared with ascorbic acid uptake, DHA produced a much greater ascorbic acid accumulation in dog erythrocytes. Both dog erythrocytes showed a concentration dependence of DHA uptake, and cellular ascorbic acid concentrations were significantly higher in high-GSH cells than in normal-GSH cells. Glucose and cytochalasin B inhibited DHA uptake. This suggests that DHA enters dog erythrocytes predominantly by the facilitated glucose transporter, particularly by the Glut 1 glucose transporter. The rate of glucose uptake was quite similar in the two types of cells. Compared with normal-GSH cells, high-GSH cells were more resistant to oxidative stress induced by high concentration of DHA. As a rapid entry of DHA inflicts on cells a heavy demand for GSH for its reduction to ascorbic acid, high-GSH cells containing a larger reserve of GSH have an advantage over normal-GSH cells in both ascorbic acid accumulation and resisting oxidative stress produced by DHA.     

Enzyme-linked Immunosorbent Assay of Pro-gastrin-releasing Peptide for Small Cell Lung Cancer Patients in Comparison with Neuron-specific Enolase Measurement

Our previous study demonstrated that pro-gastrin-releasing peptide(31–98), or ProGRP, is a specific tumor marker in patients with small cell lung carcinoma (SCLC). Using a newly developed, highly sensitive enzyme-linked immunosorbent assay (ELISA) for ProGRP, we analyzed 1,446 samples including those obtained from 478 lung cancer patients to evaluate the clinical usefulness of this ELISA. Several properties indicated that ProGRP is a useful tumor marker for SCLC. First, ProGRP was specifically elevated in SCLC patients. In non-SCLC patients and patients with non-tumorous lung diseases, its serum level was very rarely elevated. Secondly, ProGRP was a reliable marker, in terms of the marked elevation of serum ProGRP levels in SCLC patients. Thirdly, serum ProGRP levels were elevated in SCLC patients even at a relatively early stage of this disease. Fourthly, changes in the serum ProGRP level showed an excellent correlation with the therapeutic responses in SCLC patients. Neuron-specific enolase (NSE) is accepted as a tumor marker of SCLC patients. With the aim of comparing ProGRP and NSE as tumor markers for SCLC patients, we measured serum NSE levels in all samples collected in the present study. We found that ProGRP was superior to NSE in terms of sensitivity, specificity and reliability. Therefore, we consider that ProGRP can play a major role as a clinical tumor marker for SCLC patients.     

Diazepam reduces both arterial blood pressure and muscle sympathetic nerve activity in human

It is known that benzodiazepines have a hypotensive effect, but the mechanism has not been well elucidated yet. To clarify whether this effect is due to central or peripheral mechanism, we administered 5 mg of diazepam or saline intravenously to healthy volunteers and assessed the change in blood pressure, heart rate, muscle sympathetic nerve activity and heart rate variability. After diazepam administration, systolic and mean blood pressure decreased significantly. Muscle sympathetic nerve activity was also significantly reduced but heart rate did not change, whereas the variables of spectral analysis of heart rate variability did not show significant change. We concluded that the hypotensive effect of diazepam in human is mainly due to the central mechanism.     

Patterns of C-heterochromatin and telomeric DNA in two representative groups of small apes,the genera <Emphasis Type="Italic">Hylobates</Emphasis> and <Emphasis Type="Italic">Symphalangus</Emphasis>

The course of chromosome evolution in small apes is still not clear, though painting analyses have opened the way for elucidating the puzzle. Even the C-banding pattern of the lar-group of gibbons (the genus Hylobates) is not clarified yet, although our previous studies suggested that lar-group gibbons have a unique C-banding pattern. We therefore made observations to establish C-banded karyotypes of the agile gibbons included in the lar-group. The data were compared with those of siamangs (the genus Symphalangus), which carry distinctive C-bands, to determine the chromosomal patterns in each group. C-banded chromosomes of agile gibbons showed several terminal, interstitial and paracentric bands, whose patterns are specific for each chromosome, whereas the C-bands of siamangs were located only at the terminal and centromeric regions in most chromosomes. Moreover, the C-bands of agile gibbons and siamangs were shown to be G+C-rich and A+T-rich DNA, respectively, by DAPI/C-band sequential staining. Additionally, PRINS labelling with a telomere primer revealed that agile gibbons have telomeric DNA only at chromosome ends where there is no C-band (non-telomeric heterochromatin), whereas the telomeric DNA of siamangs is located in the terminal C-banded regions (telomeric heterochromatin). Although the evolutionary mechanisms in small apes are still unknown, C-banding patterns and distribution of telomeric DNA sequences should provide valuable data to deduce the evolutionary pathways of small apes.     

Studies on silicone-grafted copolyimides, 3. Synthesis of soluble polyimide/polydimethylsiloxane graft copolymer and application to separation membrane

Polyimide/polydimethylsiloxane (PI/PDMS) graft copolymer was prepared following the macromonomer method. First, 3-(3,5-diaminobenzyloxy)propyl-terminated polydimethylsiloxane (DAB-PDMS) was prepared by hydrosilylation of allyl 3,5-dinitrobenzyl ether with hydrosilyl-terminated PDMS, followed by catalytic reduction of the two nitro groups. PI/PDMS graft copolymer was synthesized by polycondensation of DAB-PDMS with 4,4′-oxydianiline (ODA) and 4,4′-(hexafluoroisopropylidene)diphthalic anhydride (6FDA), followed by chemical imidation. The average degree of polymerization of the PDMS chain was in the range between 7 and 25. The resulting graft copolymers were soluble in several organic solvents. Membranes were prepared by solvent casting, followed by heating at 220°C in vacuo, upon which the membranes became hardly soluble in organic solvents. The gas permeability coefficients of the graft copolymer membranes were much greater than those of the polyimide homopolymer and of the same order as that of a PDMS membrane. Furthermore, a selective permeation of organic liquids, e.g., tetrahydrofuran, acetone and acetonitrile, was observed in the pervaporation of aqueous organic liquids through the copolymer membrane.     

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

Activity-inducible protein Homer1a/Vesl-1S promotes redistribution of postsynaptic protein Homer1c/Vesl-1L in cultured rat hippocampal neurons

In cultured rat hippocampal neurons, overexpression of Homer1a/Vesl-1S, an inducible protein upregulated by seizure or long-term potentiation, caused a reduction of punctate distribution of a postsynaptic protein Homer1c/Vesl-1L, without significant decrease in its total amount. Clusters of F-actin were also decreased. Treatments of cells with BDNF or a proteasome inhibitor, which cause increase in the expression level of endogenous Homer1a, also resulted in the reduction of Homer1c puncta. These results indicate that the accumulation of Homer1a, either exogenously expressed or endogenously induced, caused redistribution and dispersion of postsynaptic clusters of Homer1c and F-actin, suggesting an important role of Homer1a in synaptic remodeling.     

Characterization and localization of side population cells in mouse skin

Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 fluorescence dye, has attracted attention as a method of stem cell isolation. We identified SP cells from mouse skin using the same method as from bone marrow. This population almost completely disappeared after treatment with the calcium channel blocker verapamil. SP cells were mainly localized in the epidermis, with a few in the dermis. The ratio of SP cells decreased as the mouse became older. Surface marker analysis revealed that the sorted SP cells expressed alpha6-integrin, beta1-integrin, Sca-1, keratin 14, and keratin 19, which are proliferating and progenitor cell markers, at levels higher than in non-SP cells, while they expressed E-cadherin, CD34, and CD71 at lower levels. The expression of breast cancer resistance protein 1 (BCRP1), which participates in dye efflux, was expressed at high levels at both the protein and mRNA level in sorted SP cells. Immunohistochemical analysis showed that BCRP1 was expressed in the basal layers and hair bulge regions of mouse skin. BCRP1 mRNA was found in basal layers and hair follicles of newborn skin by in situ hybridization. These results indicate that the localization of BCRP1-positive cells is compatible with that of keratinocyte stem cells. Based on the close relationship between BCRP1 and the SP cell phenotype, we conclude that keratinocyte stem cells are closely related to the SP- or BCRP1-positive cells.