Post-natal heart adaptation in a knock-in mouse model of calsequestrin 2-linked recessive catecholaminergic polymorphic ventricular tachycardia

Cardiac calsequestrin (CASQ2) contributes to intracellular Ca²⁺ homeostasis by virtue of its low-affinity/high-capacity Ca²⁺ binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation–contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2^R33Q/R33Q and of CASQ2^−/− mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2^−/− mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.     

Effects of atrial natriuretic factor on human platelet function

We examined the hypothesis that atrial natriuretic factor (ANF), a substance with known vasorelaxant activities, shares with other vasodilators the property of inhibiting platelet function. Aggregation of citrated platelet-rich plasma (PRP) from 23 healthy volunteers induced by ADP, adrenaline, arachidonic acid, collagen, gamma-thrombin, the endoperoxide analogue U-44069, serotonin, the calcium ionophore A-23187 or platelet aggregating factor was measured after incubation of PRP with ANF for 3 minutes at concentrations of 4 X 10(-9), 4 X 10(-8) and 4 X 10(-7) M or vehicle as control. ANF decreased ADP-induced aggregation significantly (P less than 0.02), but only at the highest concentration used and to a minor extent (control: 73.6 +/- 11.2%; after ANF 4 X 10(-7) M: 60.0 +/- 17.1%, mean +/- S.D., n = 39) by a selective inhibitory effect on the secondary wave; neither aggregation by all other agents tested nor thromboxane B2 generation induced by ADP and adrenaline was altered by incubation with ANF. Although ANF thus has detectable effects on ADP-induced platelet aggregation in vitro, these data suggest that ANF is unlikely to be a physiologically significant modulator of platelet function.     

Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.     

Muscle-type MM creatine kinase is specifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP ratios

Highly purified fractions of sarcoplasmic reticulum (SR) were prepared from chicken pectoralis muscles (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) and analyzed for the presence of creatine kinase (CK). Vesicles derived from longitudinal SR contained 0.703 +/- 0.428 IU of CK/mg of (SR) protein. Immunogold localization of muscle-type MM-CK on ultrathin cryosections of muscle, after removal of soluble CK, revealed relatively strong in situ labeling of M-CK remaining bound to the M band as well as to the SR membranes. In addition, purified SR vesicles were also labeled by anti-M-CK antibodies, and the peripheral labeling was similar to that observed with anti-Ca2(+)-ATPase antibodies. Only some particulate CK enzyme was released from isolated SR membranes by EDTA/low salt buffer, and CK was resistant to extraction by 0.6 M KCl. Thus, some of the MM-CK present in muscle displays strong associative behavior to the SR membranes. The SR-bound CK was sufficient to support, in the presence of phosphocreatine plus ADP, a significant portion of the maximal in vitro Ca2+ uptake rate. The ATP regeneration potential of SR-bound CK was similar to the rate of Ca2(+)-stimulated ATP hydrolysis of isolated SR vesicles. Thus, CK bound to SR may be physiologically relevant in vivo for regeneration of ATP used by the Ca2(+)-ATPase, as well as for regulation of local ATP/ADP ratios in the proximity of the Ca2+ pump and of other ATP-requiring reactions in the excitation-contraction coupling pathway.     

Polymorphism of myosin light chains. An electrophoretic and immunological study of rabbit skeletal-muscle myosins.

Antibodies specific for rabbit fast-twitch-muscle myosin LCIF light chain were purified by affinity chromatography and characterized by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA) and a gel-electrophoresis-derived assay (GEDELISA). The antibodies did not cross-react with myosin heavy chains, and were weakly cross-reactive with the LC2F [5,5'-dithio-(2-nitrobenzoic acid)-dissociated] light chain and with all classes of dissociated light chains (LC1Sa, LC1Sb and LC2S), as well as with the whole myosin, from hind-limb slow-twitch muscle. The immunoreactivity of myosins with a truly mixed light-chain pattern (e.g. vastus lateralis and gastrocnemius) correlated with percentage content of fast-twitch-muscle-type light chains. A more extensive immunoreactivity was observed with diaphragm and masseter myosins, which were also characterized, respectively, by a relative or absolute deficiency of LC1Sa light chain. Furthermore, it was found that the LC1Sb light chain of masseter myosin is antigenically different from its slow-twitch-muscle myosin analogue, and is immunologically related to the LC1F light chain. Rabbit masseter muscle from its metabolic and physiological properties and the content, activity and immunological properties of sarcoplasmic-reticulum adenosine triphosphatase, is classified as a red, predominantly fast-twitch, muscle. Therefore our results suggest that the two antigenically different iso-forms of LC1Sb light chain are associated with the myosins of fast-twitch red and slow-twitch red fibres respectively.     

Zinc opposes genotoxicity of cadmium and vanadium but not of lead

Protection by essential metals against the genotoxic effects of toxic elements is an open question. Here, human Hs27 dermal fibroblasts and B-mel melanoblasts were exposed for 10 days to (1 μM) zinc (Zn) or copper (Cu) or selenium (+ 4, Sei; + 6, Sea). Afterwards, cells were exposed for 3 days to subtoxic concentrations of lead (Pb, 100 μM) or vanadium (+ 5, V, 2 μM) or cadmium (Cd, 3 μM), slightly reducing, by themselves, cell proliferation and unaffecting cell viability and apoptosis. Genotoxic damage was evaluated by cytokinesis-block micronucleus assay (CBMN) and single cell gel electrophoresis (Comet assay, CA). CBMN and CA were preliminarly assessed following 3, 10 and 30 days of exposure to the above concentrations of Pb, V and Cd: Pb induced micronuclei (MN) formation in both Hs27 and B-mel cells, without determining direct DNA damage (as shown by CA); V did not reveal genotoxic effects on fibroblasts (as shown by CBMN and CA) but increased the frequency of MN and comets in melanoblasts; Cd induced a great number of MN and comets in fibroblasts but not in melanoblasts; all these effects did not differ after 3, 10 or 30 days of exposure to such elements so that Hs27 and B-mel cells were exposed to Pb,V and Cd for 3 days following pretreatment with (1 μM) Zn, Cu, Sei or Sea. By itself, the 10 day-exposure to (1 μM) Zn, Cu, Sei or Sea did not affect cell proliferation, viability, apoptosis and formation of MN or comets in either Hs27 or B-mel cells. Only Zn significantly reduced the Cd- and V-induced MN and comet formation in fibroblasts and melanoblasts, respectively; in these cells, however, Zn did not affect the Pb-induced MN formation. These results emphasize the role of Zn, in respect to other essential metals, in opposing the genotoxic effects of cancerogenic (Cd) or potentially cancerogenic elements (V).     

Cytokine expression and ultrastructural alterations in fresh-frozen,freeze-dried and γ-irradiated human amniotic membranes

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.     

Postoperative radiotherapy in prostate cancer: Analysis of prognostic factors in a series of 282 patients

Aim

To assess the outcomes of patients treated with postoperative RT in relation to the possible prognostic factors.

Background

Postoperative radiotherapy (RT) has been proved to reduce the risk of biochemical recurrence in high-risk prostate cancer patients. Baseline prostate specific antigen (PSA), pathological Gleason score (GS), positive surgical margins, nodal status and seminal vesicle invasion are independent predictors of biochemical relapse.

Materials and methods

The clinical records of 282 patients who underwent postoperative RT were retrospectively reviewed. The prognostic value of postoperative PSA, preoperative risk class, nodal status, pathological GS, margins status, and administration of hormonal therapy (HT) was analyzed.

Results

Postoperative RT was delivered with a median dose to the prostatic fossa of 66 Gy (range 50–72) in 1.8–2 Gy/fraction. Median follow-up was 23.1 months (range 6–119). Five-year actuarial biochemical disease-free survival (bDFS) and overall survival rates were 76% and 95%, respectively. Higher bDFS was found for patients with postoperative PSA <0.02 ng/ml (p = 0.03), low preoperative risk class (p = 0.01), pN0 (p = 0.003), GS 4–6 (p = 0.0006), no androgen deprivation therapy (p = 0.02), and irrespective of surgical margin status (p = 0.10). Multivariate analysis showed that postoperative PSA and Gleason score had a significant impact on bDFS (p = 0.039 and p = 0.05, respectively).

Conclusions

Postoperative RT with a dose of 66 Gy offers an acceptable toxicity and an optimal disease control after radical prostatectomy in patients with different risk features. A postoperative PSA >0.02 ng/ml could be considered as a prognostic factor and a tool to select patients at risk for progression.     

Microglial Phagocytosis of Dopamine Neurons at Early Phases of Apoptosis

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.