The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

TGF-beta(1) gene polymorphism in psoriasis vulgaris

Overexpression of TGF-beta(1) has been implicated in the pathology of many inflammatory diseases, including psoriasis. This study was performed to investigate the association between TGF-beta(1) single nucleotide polymorphism and susceptibility for psoriasis vulgaris. DNA from 78 patients with psoriasis vulgaris and 74 healthy volunteers was investigated. Polymorphism of TGF-beta(1) gene in codon 10 (T/C) and codon 25 (G/C) was evaluated by PCR-SSP and the results were compared between group of psoriatic patients, divided into early onset of psoriasis (type I) and late onset of psoriasis (type II) subgroups, and control healthy subjects. Frequencies in genotypes were similar between patients and control group (p >0.7), but between type I and type II psoriasis patients highly significant difference was found (p <0.0003). Higher frequency of CC/GG (intermediate producer) and TC/GG (high producer) was noted in the type I group, but the second high producer genotype (TT/GG) was more common in type II group. Also between type II psoriasis patients and healthy controls statistically significant difference was found (p <0.000001). In analyzing frequencies of carriage and alleles no significant differences were found. TGF-beta(1) gene polymorphism in codon 10 and 25 is not associated with susceptibility to psoriasis vulgaris, but may be important for the type of the disease.     

The effect of lead on photosynthesis and respiration in detached leaves and in mesophyll protoplasts of Pisum sativum

Photosynthesis and transpiration rate of detached leaves of pea (Pisum sativum L. cv. Iłowiecki) exposed to solution of Pb(NO₃)₂ at 1 or 5 mmol·dm⁻³ concentrations were inhibited. The higher concentration of this toxicant decreased photosynthesis and transpiration rates 2 and 3 times respectively, and increased respiration by about 20 %, as measured after 24 hours of treatment. Similarly to Pb(NO₃)₂, glyceraldehyde solution, an inhibitor of phosphoribulokinase, at 50 mmol·dm⁻³ concentration decreased the rates of photosynthesis and transpiration during introduction into pea leaves. The rate of dark respiration, however, remained unchanged during 2 hours of experiment. The potential photochemical efficiency of PS II (Fv/Fm) and the activity of Rubisco (EC 4.1.1.39) at 5 mmol·dm⁻³ of Pb(NO₃)₂ were lowered by 10 % and 20 % respectively, after 24 hours. Neither changes in the activity of PEPC (EC 4.1.1.31) or protein and pigment contents were noted in Pb-treated leaves. The photosynthetic activity of protoplasts isolated from leaves treated for 24 or 48 hours with Pb(NO₃)₂ at 5 mmol·dm⁻³ concentration was decreased 10 % or 25 %, whereas, the rate of dark respiration was stimulated by about 40 % and 75 %, respectively. The content of abscisic acid, a hormone responsible for stomatal closure, in detached pea leaves treated for 24 h with 5 mmol·dm⁻³ of Pb(NO₃)₂ solution was increased by about 3 times; a longer (48h) treatment led to further increase (by about 7 times) in the amount of this hormone. The results of our experiments provide evidences that CO₂ fixation in detached pea leaves, at least up to 24 hours of Pb(NO₃)₂ treatment, was restricted mainly by stomatal closure.     

Tissue-specific expression and endogenous subcellular distribution of the inositol 1,3,4,5-tetrakisphosphate-binding proteins GAP1(IP4BP) and GAP1(m)

GAP1(IP4BP) and GAP1(m) belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1(IP4BP) is primarily localised to the plasma membrane, whereas GAP1(m) appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1(m) and several Ca2+ store markers, consistent with the lack of co-localisation between GAP1(m) and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1(m) does not reside at a site where it could regulate the ability of InsP4 to release intracellular Ca2+. As GAP1(m) is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

The effects of growth hormone treatment in patients with somatotropin deficiency during their developmental age

INTRODUCTION: In Poland treatment with growth hormone of adolescent patients dates back to 1964. Till 1993 the therapy was conducted in an interrupted manner, depending on the periodic availability of the drug. The data form such forms of therapy suggested that the end height within 3rd centile was achieved only by a portion of treated patients. Since 1995 the growth hormone is used in continuous therapy, which allows to sum up the effects of the therapy, including the growth rate and end height. MATERIAL AND METHODS: A total of 117 children and adolescent of both sexes, aged 4.6 to 18.1 years, with diagnosed somatotropic or multihormonal pituitary insufficiency were included in the study. All of them were treated with growth hormone and had an analysis of growth rate and end height. RESULTS: In the first 6 months of growth hormone treatment the growth rate achieved 10.4 cm/year in boys and 10.0 in girls and showed no correlation with maturation status. In the second half of the year the growth rate declined slightly. During the remainder of the therapy the growth rate markedly declined, and this effect was most notable in girls. In 93% of patients after the end of therapy the final height was no different than the expected height. CONCLUSIONS: 1. The growth rate in first half a year of the treatment was 3 times higher than before the beginning of therapy. 2. In the second half of the first year the growth rate slightly declined. 3. In following years the growth rate declined notably. 4. The final achieved height in most of the patients does not differ from the prognosed height.     

Calcineurin and intracellular Ca2+-release channels: regulation or association?

The Ca(2+)- and calmodulin-dependent phosphatase calcineurin was reported to interact with the inositol 1,4,5-trisphosphate receptor (IP(3)R) and the ryanodine receptor (RyR) and to modulate their phosphorylation status and activity. However, controversial data on the molecular mechanisms involved and on the functional relevance of calcineurin for these channel-complexes have been described. Hence, we will focus on the functional importance of calcineurin for IP(3)R and RyR function and on the different mechanisms by which Ca(2+)-dependent dephosphorylation can affect the gating of those intracellular Ca(2+)-release channels. Since many studies made use of immunosuppressive drugs that are inhibiting calcineurin activity, we will also have to take the different side effects of these drugs into account for the proper interpretation of the effects of calcineurin on intracellular Ca(2+)-release channels. In addition, it became recently known that various other phosphatases and kinases can associate with these channels, thereby forming macromolecular complexes. The relevance of these enzymes for IP(3)R and RyR functioning will be reviewed since in some cases they could interfere with the effects ascribed to calcineurin. Finally, we will discuss the downstream effects of calcineurin on the regulation of the expression levels of intracellular Ca(2+)-release channels as well as the relation between IP(3)R- and RyR-mediated Ca(2+) release and calcineurin-dependent gene expression.