Cardiopulmonary function in bicycle racing over mountainous terrain at moderate altitude

To examine cardiopulmonary function during exercise in a mountainous region at moderate altitude, we measured cardiac frequency, oxygen consumption , and percentage arterial hemoglobin oxygen saturation (%SaO₂) before and after a bicycle race with a starting point at 638 m and finishing point at 1980 m. The time required to ascend an elevation of 10 m was prolonged with increasing altitude, and heart rate also increased with altitude. The %SaO₂ at the starting point and at the finishing point differed significantly (P<0.01). Faster cyclists exhibited higher %SaO₂ and lower , while slower cyclists exhibited a reduction in %SaO₂ and an increase in immediately after the race. The %SaO₂ recovery time was significantly correlated with the racing time (r=0.54,P<0.001). Therefore, the faster cyclists' oxygen debt upon completion of the race may be small and recovery of cardiopulmonary function may be fast, while the slower cyclists' oxygen debt may be large and recovery of cardiopulmonary function may be slow.     

Establishing apoptosis resistant cell lines for improving protein productivity of cell culture

The authors established apoptosis resistant COS–1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS–1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl–2 gene. Both bcl–2 and mock transfected COS–1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl–2 transfected cells was ninefold of that of the mock transfectants. Both bcl–2 and mock transfectants were further transfected with the vector pcDNA- containing SV40 ori and immunoglobulin gene for transiently expressing protein. The bcl–2 expressing COS–1 cells produced more protein than the mock transfected COS–1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl–2 gene. Both bcl–2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl–2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag–1 with bcl–2 improved survival of hybridoma 2E3 cells more than bcl–2 expression alone. The bag–1 and bcl–2 coexpressing cells produced more IgG than the the cells expressing bcl–2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

Possible participation of glucocorticoid in elevation of 3',5'-cyclic AMP levels through inhibition of cyclic AMP phosphodiesterase.

Administration of prednisolone and cholate to rats elevated levels of cAMP (adenosine 3',5'-cyclic monophosphate) by 1.5- to 2.0-fold. Compounds such as prednisolone, hydrocortisone, cholate, and deoxycholate were found to be potent inhibitors of partially purified cAMP phosphodiesterase prepared from rat liver. Kinetic analysis showed that the prednisolone inhibition was noncompetitive with a Ki of 8.9 x 10(-4) M. These results suggest that in addition to increasing DNA-dependent RNA polymerase activity in vivo, a large application of glucocorticoid may incur elevation of intracellular cAMP levels.     

Enzyme systems involved in phosphorylation and dephosphorylation of two endogenous phosphoproteins in neuronal membranes.

Adenosine 3′:5′-monophosphate-dependent protein kinase and phosphoprotein phosphatases were solubilized by Triton X-100, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes, and partially purified. The properties of these partially purified enzymes were studied using two substrates, Protein I and Protein II, prepared from the synaptic membrane fraction, as well as the substrates protamine and histone. The results suggest that the phosphorylation of Protein I and Protein II, as well as protamine and histone, are catalyzed by a single species of cAMP-deperident protein kinase. Thus, a single peak of protein kinase activity was observed, upon DEAE-cellulose hromatography of the Triton X-100 extract of the synaptic membrane preparation, which catalyzed the phosphorylation of all four substrate proteins. Moreover, the activity of this partially purified protein kinase toward the various substrate proteins was altered in a parallel fashion, either when the protein kinase preparation was subjected to heat inactivation or pH inactivation, or when the enzyme was assayed in the presence of various concentrations of cyclic nucleotides or of a protein kinase modulator. The individual protein substrates acted as competitive inhibitors with respect to one another. Upon sucrose density gradient centrifugation, the protein kinase activity toward the various substrates sedimented as a single peak. Finally, the relative specific activities toward the various substrates did not change significantly during a 2000-fold purification of the enzyme. In contrast to these observations with protein kinase, two peaks of protein phosphatase activity, with markedly different specificities toward Protein I and Protein II, were found upon DEAE-cellulose and Bio-Gel P-200 column chromatography of the Triton X-100 extract of the synaptic membrane fractions. One peak catalyzed the dephosphorylation of Phosphoprotein I but not of Phosphoprotein II, whereas the other peak catalyzed the dephosphorylation of Phosphoprotein II but not of Phosphoprotein I. The dephosphorylation of Phosphoprotein I by Phosphoprotein I phosphatase was not affected by adenosine 3':5'-monophosphate, whereas the dephosphorylation of Phosphoprotein II by Phosphoprotein II phosphatase required the presence of this nucleotide. Moreover, the two phosphatases differed from one another with respect to Stokes' radius as well as sedimentation coefficient.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.     

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.