Necrotic volume increase and the early physiology of necrosis

Whether a lethally injured mammalian cell undergoes necrosis or apoptosis may be determined by the early activation of specific ion channels at the cell surface. Apoptosis requires K+ and Cl- efflux, which leads to cell shrinking, an active phenomenon termed apoptotic volume decrease (AVD). In contrast, necrosis has been shown to require Na+ influx through membrane carriers and more recently through stress-activated non-selective cation channels (NSCCs). These ubiquitous channels are kept dormant in viable cells but become activated upon exposure to free-radicals. The ensuing Na+ influx leads to cell swelling, an active response that may be termed necrotic volume increase (NVI). This review focuses on how AVD and NVI become conflicting forces at the beginning of cell injury, on the events that determine irreversibility and in particular, on the ion fluxes that decide whether a cell is to die by necrosis or by apoptosis.     

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the referred kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.     

Changes in embryo production results and ovarian recrudescence during the acclimatisation to the semiarid tropics of embryo donor Holstein-Friesian cows raised in a temperate climate

Pregnant Holstein-Friesian (HF) heifers were transported from central Europe (defined as temperate conditions) to north-eastern Brazil (defined as tropical, semiarid conditions). They were kept in open-sided pens with a hard floor, a roof for shade and sprinkled with water for 10 min every hour if ambient temperature exceeded 30 degrees C. Their diet was balanced to meet nutritional requirements and they were fed twice daily. Control animals were randomly chosen first and second lactation animals located on a farm 25 km away and receiving similar management. Imported animals were superovulated in 1996 (n=63) and 1997 (n=96), compared to 38 and 45 cows in the control herd. The variates recorded were: the interval post-partum to first oestrus; changes in ovarian size and activity; responses to superovulation; and, embryo quality.The average daily milk yields of the imported cows were 20.0 and 23.3 l in 1996 and 1997, respectively compared to 22.1 l throughout the experiment for cows in the control herd.The post-partum anoestrus interval in the imported cows were 112.1+/-30.5 days in 1996 compared to 55.0+/-18.0, 48.2+/-12.0 and 42.6+/-10.7 days in 1997 for control cows. The size and functionality of the ovaries was lowest for the imported animals in 1996 but did not differ between other group-year combinations. These animals also had a lower superovulatory response in 1996 than control cows in terms of the number of ovulations (6.4+/-4.3 versus 13.6+/-5.9, P<0.05) and good quality embryos (1.2+/-0.9 versus 4.4+/-2.1, P<0.05). The two groups of cows did not differ in respect of these characters in the second year of the study.The imported cows had lower reproductive efficiency and responses to superovulation in their first year in their new environment. A period of approximately 1.5 years is required for full adaptation.     

Fertilizing characteristics of bovine sperm with flattened or indented acrosomes

Frozen semen from a control bull (C: 89% morphologically normal sperm) and two bulls with acrosomal defects (K1: 92% flattened acrosomes; K2: 82% indented acrosomes) were used to investigate the fertilizing ability of bull sperm with flattened or indented acrosomes. In experiment 1, frozen-thawed sperm were evaluated for acrosomal integrity with fluorescent microscopy. In experiment 2, proteolytic activity of the acrosomal contents of sperm was evaluated through a gelatin digestion assay. In experiment 3, an IVF test system was used to determine the ability of sperm with flattened or indented acrosomes to bind to bovine oocytes and penetrate the zona pellucida. In experiment 4, IVM zona-free bovine oocytes (ZFO) were fertilized and examined to evaluate sperm chromatin decondensation. In experiment 1, bulls K1 and K2 had a lower proportion of sperm with intact acrosomes (0 and 13.6 +/- 4.5%, respectively) than bull C (30.2 +/- 5.6%) after 2h of incubation. In experiment 2, the proportion of sperm with proteolytic activity, as indicated by gelatin digestion around sperm heads, did not differ among bulls (C: 55%, n=410; K1: 43%, n=426; K2: 48%, n=324). In experiment 3, a lower proportion of sperm with flattened (K1) or indented acrosomes (K2) bound to oocytes than sperm from the control bull, C. The percentage of zona penetrated (55%, n=20; 13%, n=23; 4%, n=25) and the mean (+/- S.E.M.) number of sperm penetrating these zona pellucida (19.7 +/- 2.5; 6.9 +/- 1.0; and 2.6 +/- 0.5) was higher (P<0.05) for bull C than for bulls K1 or K2, respectively. In experiment 4, the percentage of ZFO penetrated (95%, n=20; 52%, n=30; 30%, n=33) and the mean (+/- S.E.M.) number of sperm with chromatin decondensation (7.8 +/- 1.6; 0.8 +/- 0.2; and 0.3 +/- 0.1) were also higher (P<0.05) for the control bull, C than for bulls K1 or K2, respectively. Results suggest that although sperm with the flattened or indented acrosomes had a tendency to undergo spontaneous acrosome reaction on incubation after thawing, the proteolytic activity of the acrosomal contents appeared to be normal. Sperm with the flattened or indented acrosomes also appeared to have a reduced ability to fuse with oolemma as demonstrated by confocal microscopy. This would impair the ability to penetrate ooplasm and undergo sperm chromatin decondensation.     

Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain

Prenatal stress greatly influences the ability of an individual to manage stressful events in adulthood. Such vulnerability may result from abnormalities in the development and integration of forebrain dopaminergic and glutamatergic projections during the prenatal period. In this study, we assessed the effects of prenatal stress on the expression of selective dopamine and glutamate receptor subtypes in the adult offsprings of rats subjected to repeated restraint stress during the last week of pregnancy. Dopamine D₂-like receptors increased in dorsal frontal cortex (DFC), medial prefrontal cortex (MPC), hippocampal CA₁ region and core region of nucleus accumbens (NAc) of prenatally stressed rats compared to control subjects. Glutamate NMDA receptors increased in MPC, DFC, hippocampal CA₁, medial caudate-putamen, as well as in shell and core regions of NAc. Group III metabotropic glutamate receptors increased in MPC and DFC of prenatally stressed rats, but remained unchanged in all other regions examined. These results indicate that stress suffered during the gestational period has long lasting effects that extend into the adulthood of prenatally stressed offsprings. Changes in dopamine and glutamate receptor subtype levels in different forebrain regions of adult rats suggest that the development and formation of the corticostriatal and corticolimbic pathways may be permanently altered as a result of stress suffered prenatally. Maldevelopment of these pathways may provide a neurobiological substrate for the development of schizophrenia and other idiopathic psychotic disorders.     

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.     

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.     

Evaluation of a Western blot test,Helico blot 2.1, in the diagnosis of Helicobacter pylori infection in a pediatric population

Background. Noninvasive diagnostic tests are useful as screening tools for Helicobacter pylori infection in pediatric populations. The aim of this study was to evaluate performance of the immunoblot assay, Helico Blot 2.1, for the diagnosis of H. pylori infection in symptomatic children. Materials and Methods. Immunoblot assay was used for detection of IgG antibodies to specific H. pylori proteins and to a recombinant H. pylori antigen, CIM marker. The study was performed on sera collected from 134 symptomatic, untreated children (mean age, 9.1 ± 3.2 years; range, 1–14 years). H. pylori infection status was determined by culture, histology and rapid urease test. Results. Immunoblot assay yielded a positive result in 71 of the 72 infected patients (sensitivity 98.6%) and in eight of the 62 noninfected ones (specificity 87.1%). The predictive values for a positive and a negative result were 89.9% and 98.2%, respectively. The performance of the CIM band alone, as a marker for H. pylori infection status, was also evaluated. This band was present on the blot of 71 infected patients and on four of the 62 H. pylori‐negative patients. The sensitivity, specificity, PPV and NPV of the CIM antigen were 98.6%, 93.5%, 94.7% and 98.3%, respectively. Conclusions. The immunoblot assay Helico Blot 2.1 is a suitable noninvasive test for the serodiagnosis of H. pylori infection in children. The good level of performance demonstrated by the novel recombinant antigen CIM suggests it may be a useful contribution to the qualitative and quantitative performance of the Helico Blot 2.1 in pediatric populations.     

Molecular pharmacological dissection of short- and long-term memory

1. It has been discussed for over 100 years whether short-term memory (STM) is separate from, or just an early phase of, long-term memory (LTM). The only way to solve this dilemma is to find out at least one treatment that blocks STM while keeping LTM intact for the same task in the same animal.2. The effect of a large number of treatments infused into the hippocampus, amygdala, and entorhinal, posterior parietal or prefrontal cortex on STM and LTM of a one-trial step-down inhibitory avoidance task was studied. The animals were tested at 1.5 h for STM, and again at 24 h for LTM. The treatments were given after training.3. Eleven different treatments blocked STM without affecting LTM. Eighteen treatments affected the two memory types differentially, either blocking or enhancing LTM alone. Thus, STM is separate from, and parallel to the first hours of processing of, LTM of that task.4. The mechanisms of STM are different from those of LTM. The former do not include gene expression or protein synthesis; the latter include a double peak of cAMP-dependent protein kinase activity, accompanied by the phosphorylation of CREB, and both gene expression and protein synthesis.5. Possible cellular and molecular events that do not require mRNA or protein synthesis should account for STM. These might include a hyperactivation of glutamate AMPA receptors, ribosome changes, or the exocytosis of glycoproteins that participate in cell addition.     

Evaluation of the impact of Bacillus thuringiensis serovar israelensis and Temephos,used for the control of Simulium (Chirostilbia) pertinax Kollar, 1832 (Diptera,Simuliidae) on the associated entomofauna,Paraty, state of Rio de Janeiro,Brazil

The study was set up to evaluate the impact of two commercial larvicide formulations, Bacillus thuringiensis serovar israelensis base (Bti) at 15 ppm/1 min and temephos at 0.03 ppm of active ingredient, used to control Simulium pertinax populations, on associated non-target entomofauna occupying the same breeding sites. The experiments were carried out on the Pedra Branca and Muricana rivers, on the slopes of Serra do Mar massif, municipality of Paraty, state of Rio de Janeiro, Brazil. Bti was applied to the river Pedra Branca and temephosto theriver Muricana. On both rivers, treatment and control sections were labeled as such, each one with two observation posts: slow moving water and fast water regions respectively. Artificial substrata was used to evaluate the abundance of associated entomofauna. Attached immature stages of arthropods were removed from both of its surfaces fortnightly. Were collected, from the two rivers, 28 477 specimens of the entomofauna associated with S. pertinax. The families Hydropsychidae, Chironomidae, Bactidae, Simuliidae, Blephariceridae and Megapodagrionidae were represented. These was an impact of temephos on the entomofauna associated with S. pertinax only in Simuliidae and Chironomidae, and to Bti only in Simuliidae. However, the reduction in their numbers was not statistically significant.