Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.     

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.     

Appearance of retrogradely labeled neurons in the rat superior cervical ganglion after injection of wheat-germ agglutinin-horseradish peroxidase conjugate into the contralateral ganglion

Summary Injection of wheat-germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the superior cervical ganglion (SCG) of the rat results in accumulation of WGA-HRP in sympathetic postganglionic neurons in the contralateral SCG. The sympathetic pathways involved and the mechanism underlying the labeling were investigated. The labeling in neurons in the contralateral SCG was apparent 6 h after injection and increased in intensity with longer survival times. The number of labeled neurons reached 1300 at 72 h after the injection. Transection of the external (ECN) or internal carotid nerves (ICN) resulted in considerable reduction in the number of labeled neurons. Combined transection of both ECN and ICN virtually eliminated labeling in the contralateral SCG. This provides strong evidence that these two nerves are the major pathways for WGA-HRP transport out of the SCG. No labeling was observed in the contralateral SCG following injection of horseradish peroxidase (HRP). Therefore, it seems unlikely that a direct nerve connection exists between the bilateral ganglia. Instead, the labeling of contralateral SCG neurons appears to depend on the transneuronal transport capacity of WGA-HRP, which conveys the marker in an anterograde direction along the postganglionic fibers to terminals in sympathetic target organs, and then delivers it transneuronally to contralateral SCG neurons. We suggest that the sympathetic nerve fibers originating in the bilateral SCGs run intermingled and are in close contact in their peripheral target organs.     

Effect of high-intensity endurance training on isokinetic muscle power

The purpose of this study was to determine the effects of high-intensity endurance training on isokinetic muscle power. Six male students majoring in physical-education participated in high intensity endurance training on a cycle ergometer at 90% of maximal oxygen uptake (VO2max) for 7 weeks. The duration of the daily exercise session was set so that the energy expenditure equalled 42 kJ.kg-1 of lean body mass. Peak knee extension power was measured at six different speeds (30 degrees, 60 degrees, 120 degrees, 180 degrees, 240 degrees, and 300 degrees.s-1) with an isokinetic dynamometer. After training, VO2max increased significantly from mean values of 51.2 ml.kg-1.min-1, SD 6.5 to 56.3 ml.kg-1.min-1, SD 5.3 (P less than 0.05). Isokinetic peak power at the lower test speeds (30 degrees, 60 degrees and 120 degrees.s-1) increased significantly (P less than 0.05). However, no significant differences in muscle peak power were found at the faster velocities of 180 degrees, 240 degrees, and 300 degrees.s-1. The percentage improvement was dependent on the initial muscle peak power of each subject and the training stimulus (intensity of cycle ergometer exercise).     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Protection of mice against Sendai virus pneumonia by non-neutralizing anti-F monoclonal antibodies

Nine monoclonal antibodies (MAbs) directed to F protein of Sendai virus were obtained and characterized for their protective ability against Sendai virus infection in mice. None of the MAbs showed hemagglutination-inhibition (HI), hemolysis-inhibition (HLI), or neutralization (NT) activities in vitro when assayed by standard methods. Some of the MAbs, however, showed complement-requiring NT (C-NT) and complement-requiring hemolysis (C-HL) activities when assayed in the presence of complement. Passive immunization experiments revealed that the MAbs with higher C-NT and C-HL activities showed protective activity against Sendai virus pneumonia in mice, and that some MAbs with IgG1 isotype having neither C-NT nor C-HL activity also showed the protective activity. Digestion of the MAbs with pepsin which split immunoglobulin molecules into F(ab')2 and Fc fragments greatly suppressed the protective activity. These results suggest that not only complement-mediated immunological responses such as immune virolysis but also antibody-dependent cellular cytotoxicity (ADCC) and/or immune phagocytosis, in which complement system is not necessarily involved, play an important role in the protection of mice from Sendai virus infection.     

Growth analysis ofQuercus serrata seedlings withinMiscanthus sinensis grass canopies differing in light availability

The effects of different light regimes on the survival, growth and morphology ofQuercus serrata seedlings were studied in canopies ofMiscanthus sinensis. The seedlings of various ages (0–3 yr) were grown in three light regimes: under a denseM. sinensis canopy (TG plot) receiving 2.5%–8.7% of full sunlight, under a relatively sparse canopy (SG plot) receiving 3.8%–16.1% of light and in an adjacent open site (NG plot). There was a little difference in the survival ofQ. serrata seedlings among the three plots. Height and diameter of stem and total leaf area of the seedlings were significantly lower in the shadier plots. However, the first (bottom) flush of the stem was significantly longer in the TG plot than in the NG and SG plots. Total dry weights of individual 1- and 2-yr-oldQ. serrata seedlings in the TG plot were reduced to about one-twelfth of those in the NG plot. Although the relative proportion in dry weight of each organ did not differ significantly among the plots, leaf area ratio, specific leaf area and stem height per unit dry weight were significantly higher in shadier plots. The leaf area per unit stem height was increased considerably in the sunnier plots.     

Effects of hydrocortisone and aminophylline on plasma leukotriene C4 levels in patients during an asthmatic attack

To study the role of leukotriene C4(LTC4) and the effect of hydrocortisone and aminophylline on plasma LTC4 levels in patients with asthmatic attacks, we measured LTC4 in plasma of 18 asthmatics during a wheezing attack and of 7 normal subjects. Blood samples were obtained before and after treatment with aminophylline and/or hydrocortisone injections. We extracted LTC4 using a Sep-Pak C18 cartridge for the measurement of LTC4 by radioimmunoassay. The plasma levels of immunoreactive LTC4 (i-LTC4) of the normal subjects were 142 +/- 25 pg/ml (n = 7), while those of nonatopic type asthmatic patients with wheezing attacks were 208 +/- 68 pg/ml (n = 15) (p less than 0.01). Before and after treatment with both hydrocortisone succinate (100 mg) and aminophylline (250 mg), 6 asthmatic patients with wheezing attacks had a mean plasma level of i-LTC4 181 +/- 24 and 132 +/- 18 pg/ml (p less than 0.01), respectively. On the other hand, the treatment with aminophylline 250 mg alone increased the i-LTC4 levels from 178 +/- 19 pg/mg to 213 +/- 16 pg/mg (n = 6)(p less than 0.05), while treatment with hydrocortisone succinate 100 mg decreased the i-LTC4 level 0.05 from 284 +/- 99 pg/ml to 249 +/- 85 pg/ml (n = 4)(p less than 0.05). In conclusion, the present study shows that the i-LTC4 level in venous blood of patients with asthmatic attacks is decreased significantly by treatment with hydrocortisone succinate.     

Ornithine decarboxylase antizyme in kidneys of male and female mice.

Antizyme, a protein inhibitor of ornithine decarboxylase (ODC), was shown to be induced in mouse kidney by repeated injection of putrescine. Antizyme was also present as a complex with ODC in the kidney of untreated mouse. The amount of the renal ODC-antizyme complex was 3-fold higher in male mice than in female mice. On the contrary, the proportion of ODC present as a complex with antizyme was 24-fold higher in females than in males, and the decay of renal ODC activity after cycloheximide treatment was about 5-fold more rapid in females than in males. Administration of testosterone to female mice, a procedure known to prolong the half-life of renal ODC, increased both ODC activity and the content of ODC-antizyme complex, but decreased the antizyme/ODC ratio in the kidney. These results are consistent with the previous observation in HTC cells that the decay rate of ODC activity in the presence of cycloheximide correlated well with the proportion of ODC present as a complex with antizyme, suggesting the ubiquitous role of antizyme in ODC degradation.