Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.     

Digital multi-filter auditory prosthesis: study of its efficiency in function of number of filters and their programming

A new method to determine a rate constant and total reaction product using data of reaction product kinetics is proposed. The method is based on the solution of the system of irrational equations (for special case), which describes such processes. This method was used to determine the reaction rate constant of interaction of some monoclonal antibodies with the antigens immobilized on immunological plates.     

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.     

Involvement of the esophagus in the cramp-fasciculation syndrome

A 35-year-old male patient presented with symptoms of a cramp-fasciculation syndrome, but also reported difficulties swallowing. Esophageal manometry showed spontaneous nonperistaltic contractions, pathologically increased amplitudes and duration of the contractile complexes, and an asynchronous propagation. Electromyographic evidence of fasciculations in the sternocleidomastoid and pectoralis muscles was found. Apparently all types of peripheral motor fibers can be involved in this heterogeneous syndrome, including cranial motor nerves, the vagal nerve, and enteric motor fibers of the gastrointestinal tract.     

CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells

We hypothesized that human hematopoietic cells displaying a CD34+, kit-, rhodamine123(low) phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123(low) cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33-, CD38-, CD20-, and glycophorin A-. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 x 10(3) candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.     

Anti-D in a D-positive renal transplant patient

PURPOSE: We report a case of postoperative reparalysis in the recovery room, following nebulized epinephrine. The patient was pharmacologically reversed with edrophonium after paralysis with rocuronium. CLINICAL FINDINGS: A 12-yr-old girl developed postoperative reparalysis following the intraoperative administration of rocuronium. A total of 0.92 mg.kg-1 rocuronium was administered. After surgery, pharmacological reversal was achieved with 20 mg edrophonium with 0.15 mg atropine sulfate iv 35 min after the last administration of rocuronium. Muscular relaxation was monitored using an ulnar peripheral nerve stimulator (PNS). After reversal, a full train-of-four and sustained tetanus at 50 Hz were present. In the recovery room, following nebulized epinephrine, the patient became apneic. The patient was paralyzed and an ulnar PNS demonstrated only one faint twitch. The paralysis was reversed with 1.5 mg neostigmine with 0.3 mg glycopyrrolate. CONCLUSION: Postoperative reparalysis following rocuronium may be a cause of postoperative respiratory distress. The definitive diagnosis is made using PNS and observing the response to pharmacological reversal. Nebulized epinephrine may have a previously undescribed role in the development of postoperative reparalysis.     

In vitro and in vivo inhibitory actions of morin on rat brain phosphatidylinositolphosphate kinase activity

Phosphatidylinositol-4,5-bisphosphate occupies a central role in signal transduction and in cellular transformation. Phosphatidylinositol-4,5-bisphosphate is produced by the enzymatic phosphorylation of phosphatidylinositol-4-phosphate by phosphatidylinositolphosphate kinase (EC 2.7.1.68). Inhibition of this enzyme might conceivably lowers the cellular pool of phosphatidylinositol-4,5-bisphosphate, thus constituting a feasible control point in regulating signal transduction and cellular transformation. Morin, a plant flavonoid, was demonstrated to exhibit in vitro inhibitory action on phosphatidylinositolphosphate kinase extracted from rat brain. This inhibition of enzymatic activity was found to be dose-dependent, with an IC50 value of approximately 10 microM morin. Lineweaver-Burk transformation of the inhibition data indicates that inhibition was competitive with respect to ATP. The Ki was calculated to be 5.15 x 10(-6) M. Inhibition was uncompetitive with respect to phosphatidylinositol-4-phosphate. The Ki was determined to be 0.94 x 10(-5) M. Administration of morin to rats led to a decrease in phosphatidylinositolphosphate kinase activity in brain extracts. This in vivo action of morin was found to be dose-dependent and time-dependent. These effects of morin on rat brain phosphatidylinositolphosphate kinase activity are discussed in relation to the other reported biological actions of this flavonoid.     

Captopril modifies gene expression in hypertrophied and failing hearts of aged spontaneously hypertensive rats

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.     

Idiopathic thrombocytopenic purpura in a liver transplant recipient with previous primary biliary cirrhosis

The loss of immunotolerance has been implicated in the pathogenesis of both primary biliary cirrhosis (PBC) and idiopathic, immune-mediated thrombocytopenic purpura (ITP). An association between these two autoimmune diseases has been well described. We describe a 41-year-old woman in whom ITP developed 457 days after liver transplantation for PBC while receiving immunosuppressive medications sufficient to maintain allograft function. Our case report, the first to describe post-transplant ITP in association with PBC, demonstrates the persistence of the underlying immune dysregulation of PBC after transplantation. The practice of decreasing the dosage of immunosuppressive medication to maintenance levels after transplantation may unmask the effects of this defect in immunotolerance.     

Mechanisms underlying the chronotropic effect of angiotensin II on cultured neurons from rat hypothalamus and brain stem

The chronotropic effect of angiotensin II (Ang II) was studied in cultured neurons from rat hypothalamus and brain stem with the use of the patch-clamp technique. Ang II (100 nM) increased the neuronal spontaneous firing rate from 0.8 +/- 0.3 (SE) Hz in control to 1.3 +/- 0.4 Hz (n = 7, P < 0.05). The amplitude of threshold stimulation was decreased by Ang II (100 nM) from 82 +/- 4 pA to 62 +/- 5 pA (n = 4, P < 0.05). These actions of Ang II were reversed by the angiotensin type 1 (AT1) receptor antagonist losartan (1 microM). In the presence of tetrodotoxin, Ang II (100 nM) significantly increased the frequency and the amplitude of the Cd2+-sensitive subthreshold activity of the cultured neurons. Ang II also stimulated the subthreshold early afterdepolarizations (EADs) to become fully developed action potentials. Similar to the action of Ang II, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) increased the firing rate from 0.76 +/- 0.3 Hz to 2.3 +/- 0.5 Hz (n = 6, P < 0.05) and increased the neuronal subthreshold activity. After neurons were intracellularly dialyzed with PKC inhibitory peptide (PKCIP, 5 microM), PMA alone, Ang II alone, or PMA plus Ang II no longer increased the action potential firing initiated from the resting membrane potential level. However, superfusion of PMA plus Ang II or Ang II alone increased the number of EADs that reached threshold and produced action potentials even in the presence of PKCIP (5 microM, n = 4). The actions of Ang II could also be mimicked by depolarizing pulse and K+ channel blockers (tetraethylammonium chloride or 4-aminopyridine). These results indicate that Ang II by activation of AT1 receptors increases neuronal excitability and firing frequency, and that this may involve both PKC dependent and -independent mechanisms.