Ceramide selectively decreases tau levels in differentiated PC12 cells through modulation of calpain I

Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated PC12 cells with the cell-permeable ceramide derivative N-acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsphingosine (C6). In addition, C2 treatment increased the levels of a calpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, alpha-actin and alpha-tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of PC12 cells with C2 enhanced calcium-stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane-permeable calpain-selective fluorescence probe N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin . This activity increase was not due to a direct effect of C2 on calpains, because C2 did not alter the activities of purified calpain I or II. In addition, C2 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain-specific inhibitor N-carbobenzoxy-L-leucyl-L-leucyl-L-tyrosine diazomethyl ketone blocked the C2-induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramide-mediated changes in normal and pathological neuronal processes.     

Is it possible to perform immediate cardiac assist using untrained latissimus dorsi muscle in a work-rest regimen?

The authors investigated what contractile force (CF) could be obtained from unconditioned latissimus dorsi muscle immediately after mobilization and for the 2 week vascular period of recovery. Latissimus dorsi muscle mobilization was performed on seven adult (4 experimental and 3 control) sheep leaving only the pedicle and the peripheral muscle intact. Telectronics stimulators (Myostim 7220; Teletronics Pacing Systems, Inc, Englewood, CO) were implanted. Immediately after mobilization 11-35% of the initial CF was lost. A 30 min fatigue test was performed 1 hr after mobilization (20 g/kg preload, 10 V, 10 Hz, 15 BPM, 6 impulses per burst) using a 1 min work-1 min rest regimen. Two sheep lost 2-12% of initial CF; two increased CF by 14-24%. At the end of the fatigue test, CF consisted of 74-89% of immobilized CF. Electrical stimulation training of the muscle was then initiated with the following regimen in the experimental animals only: 15 BPM, single impulses, 5 V, 10 Hz. Every day the muscle was exercised using a work-rest regimen to mimic cardiac assist, starting with 20 min on day 2, and increasing by 2 min per day until a total of 50 min was reached on day 16. All animals were retested for CF using a 42 min fatigue test on days 6, 11, and 16. On day 6, there was no fatigue evident in the experimental group during the 42 min test. CF after testing was 59-81% (mean 67%) of initial data. In the control group (animals with no electrical stimulation training protocol), CF decreased by 11% (from 64 to 53%). On day 11, there was no fatigue evident in the experimental group; CF in all animals increased by 2-8%. On day 16, there was also no fatigue evident in the experimental group; CF increased by 0-9%. An additional 20 min of continuous contraction (15 BPM) fatigue testing was performed on the muscle without rest between the tests. No fatigue was evident at the end of testing. Light microscopic analysis of latissimus dorsi muscle biopsy specimens taken on the days of testing showed no evidence of necrotic damage. Our investigations suggest that it may be possible to start muscle transformation immediately after mobilization and use the untrained latissimus dorsi muscle for cardiac assist immediately after surgery for short periods.     

The phylogeny of the wrist

BACKGROUND: Long-term results on LASIK are not available to date. We therefore evaluated the predictability, stability and complication rate after LASIK in moderate--to-high myopia. PATIENTS AND METHODS: We treated 70 eyes (41 patients) using the Automatic Corneal Shaper and the Keracor 116 excimer laser. Patients were followed for 1, 6, 12 and 24 months. Spectacle refraction, visual acuity, rate of retreatment, and patient satisfaction were evaluated. RESULTS: At 24 months the results were as follows: Myopia -5 to -9.9 D (n = 18): 94% within 1 D; regression between 1 and 12 (12 and 24) months > 1 D in 6% (6%); uncorrected acuity 20/40 or better in 83%; no loss of 2 ore more lines of visual acuity; 89% highly satisfied. Myopia -10 to -14.9 D (n = 12): 88% within 1 D; regression between 1 and 12 (12 and 24) months > 1 D in 20% (0%); uncorrected acuity 20/40 or better in 72%; 4% lost 2 or more lines of visual acuity; 96% highly satisfied. Myopia -15 to -29 D (n = 22): 33% within 1 D; regression between 1 and 12 (12 and 24) months > 1 D in 41% (18%); uncorrected acuity 20/40 or better in 7%; no loss of 2 or more lines of visual acuity; 67% highly satisfied. CONCLUSION: LASIK is an accurate, effective and stable procedure for correcting myopia of -5 to -10 D. Results are less precise in myopia up to -15 D, and some visual loss occurs in a number of patients. In myopia > -15 D, results are not satisfactory because of poor accuracy and low stability.     

Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.     

DNA sequence recognition by bis-linked netropsin and distamycin derivatives

The dihydropyridine receptor (DHPR), a voltage-gated L-type Ca2+ channel, and the Ca2+ release channel/ryanodine receptor isoform-1 (RyR1) are key molecules involved in skeletal muscle excitation-contraction coupling. We have reported age-related decreases in the level of DHPR expression in fast- and slow-twitch muscles from Fisher 344 cross Brown Norway (F344BNX) rats (Renganathan, Messi and Delbono, J. Membr. Biol. 157 (1997) 247-253). Based on these studies we postulate that excitation-contraction uncoupling is a basic mechanism for the decline in muscle force with aging (Delbono, Renganathan and Messi, Muscle Nerve Suppl. 5 (1997) S88-92). In the present study, we extended our studies to older ages and we intended to prevent or retard excitation-contraction uncoupling by restricting the caloric intake of the F344BNX rats from 16 weeks of age. Three age groups, 8-, 18-, and 33-month old caloric restricted rats, were compared with ad libitum fed animals. The number of DHPR and RyR1 and DHPR/RyR1 ratio (an index of the level of receptors uncoupling) in skeletal muscles of 8-month and 18-month rats was not significantly different in either ad libitum fed or caloric restricted rats. However, the age-related decrease in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in 33-month old ad libitum fed rats was absent in 33-month old caloric restricted rats. These results suggest that caloric restriction prevents age-related decreases in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in fast- and slow-twitch rat skeletal muscles.