Inhibition of apoptosis by zinc: a reappraisal.

Apoptosis--or programmed cell death--is an active type of cell death, occurring in several pathophysiological conditions. One of the most important characteristics of apoptosis is that cell death is preceded by DNA fragmentation, consequent to the activation of nuclear calcium- and magnesium-dependent endonuclease(s). DNA fragmentation can be inhibited by zinc ions. By using several techniques, such as DNA agarose gel electrophoresis, cytofluorimetric analysis of DNA content and of cell cycle, 3H-thymidine incorporation and trypan blue dye exclusion test, we show that zinc, despite completely inhibiting DNA fragmentation and the consequent loss of nuclear DNA content, does not protect rat thymocytes from spontaneous or dexamethasone-induced death. Our data also suggest that DNA fragmentation, although characteristic, is not a critical event for thymocyte death of apoptotic type.     

HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death

Two hours after a single intraperitoneal injection of dexamethasone (20 micrograms/Kg b.w.) into adult male rats, a typical ladder of DNA fragments was detectable upon separation on agarose gels of DNA from thymocytes. This became maximally evident at 4 hours. Accumulation of sulfated glycoprotein-2 (SGP-2) mRNA, whose rate of expression has been associated to the processes of programmed cell death, preceded the appearance of DNA degradation, starting to increase as early as 30 min after steroid injection, and maintained higher than controls until 8 hrs; a different time course was shown by changes in the levels of beta-actin mRNA. In the spleen, under the same conditions, the SGP-2 message also increased at 30 min, prior to DNA fragmentation, but decreased thereafter below the control value.     

International Society for Biological and Environmental Repositories

Genetically altered mice are an important tool for biomedical research. Several transgenic mice have been created in which activation of the transgene results in production of β-galactosidase that can be detected by histological means. While preservation and subsequent visualization of enzyme activity in soft tissues can be complicated, it is particularly difficult in bone specimens, especially those that have been decalcified. For these studies, we examined the bones of parathyroid hormone-related peptide (PTHrP) knock-in mice in which expression of PTHrP resulted in β-galactosidase production. During the past decade, several studies have demonstrated the importance of PTHrP in bone. Thus, it is important to preserve and detect β-galactosidase enzymatic activity in bone for these studies. We demonstrate here that β-galactosidase was visualized better in slides with bone sections taken from PTHrP knock-in mice when bones were frozen and sectioned compared to bones that were embedded in plastic and sectioned using a microtome. Importantly, we were able to visualize β-galactosidase in plastic embedded bones when specimens were fixed, stained (X-gal), embedded in plastic, and then sectioned rather than being fixed, embedded in plastic, sectioned, then stained.     

Prognostic Role of Ventricular Ectopic Beats in Systemic Sclerosis: A Prospective Cohort Study Shows ECG Indexes Predicting the Worse Outcome

BackgroundArrhythmias are frequent in Systemic Sclerosis (SSc) and portend a bad prognosis, accounting alone for 6% of total deaths. Many of these patients die suddenly, thus prevention and intensified risk-stratification represent unmet medical needs. The major goal of this study was the definition of ECG indexes of poor prognosis.MethodsWe performed a prospective cohort study to define the role of 24h-ECG-Holter as an additional risk-stratification technique in the identification of SSc-patients at high risk of life-threatening arrhythmias and sudden cardiac death (SCD). One-hundred SSc-patients with symptoms and/or signs suggestive of cardiac involvement underwent 24h-ECG-Holter. The primary end-point was a composite of SCD or need for implantable cardioverter defibrillator (ICD).ResultsFifty-six patients (56%) had 24h-ECG-Holter abnormalities and 24(24%) presented frequent ventricular ectopic beats (VEBs). The number of VEBs correlated with high-sensitive cardiac troponin T (hs-cTnT) levels and inversely correlated with left-ventricular ejection fraction (LV-EF) on echocardiography. During a mean follow-up of 23.1±16.0 months, 5 patients died suddenly and two required ICD-implantation. The 7 patients who met the composite end-point had a higher number of VEBs, higher levels of hs-cTnT and NT-proBNP and lower LV-EF (p = 0.001 for all correlations). All these 7 patients had frequent VEBs, while LV-EF was not reduced in all and its range was wide. At ROC curve, VEBs>1190/24h showed 100% of sensitivity and 83% of specificity to predict the primary end-point (AUROC = 0.92,p<0.0001). Patients with VEBS>1190/24h had lower LV-EF and higher hs-cTnT levels and, at multivariate analysis, the presence of increased hs-cTnT and of right bundle branch block on ECG emerged as independent predictors of VEBs>1190/24h. None of demographic or disease-related characteristics emerged as predictors of poor outcome.ConclusionsVEBS>1190/24h identify patients at high risk of life-threatening arrhythmic complications. Thus, 24h-ECG-Holter should be considered a useful additional risk-stratification test to select SSc-patients at high-risk of SCD, in whom an ICD-implantation could represent a potential life-saving intervention.     

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

Genetic differentiation at the 3'UTR of PROS-Ag25: a cDNA homologue of Drosophila melanogaster proteasome subunit PROS-Dm25 in the malaria vector Anopheles gambiae

The Anopheles gambiae cDNA encoding the homologue of the Drosophila melanogaster proteasome PROS-Dm25 was identified and analysed in terms of nucleotide sequence, and chromosomal localisation. In the 3 untranslated region, a GA-rich sequence was mapped which was found to be widely polymorphic among taxa belonging to the A. gambiae complex.