Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Potential of some Metarhizium anisopliae isolates for control of Culex quinquefasciatus (Dipt., Culicidae)

Abstract: The potential of some isolates of Metarhizium anisopliae and Beauveria bassiana for use in the integrated management of Culex quinquefasciatus was evaluated. Metarhizium anisopliae isolate 1037 was selected in initial bioassays with a 50% lethal concentration (LC₅₀) of 1.97 × 10⁴ conidia/ml. This fungus caused higher mosquito larva mortality when applied as a conidial suspension to the surface of the water than as dry conidia, with a time to 50% lethal (LT₅₀) of 1 day compared with 3.6 days for the dry conidial application. However, results with UV- and heat-inactivated conidia did not confirm a possible role of fungal toxins in causing mortality when ingested by C. quinquefasciatus larvae. Metarhizium anisopliae did not remain active for as long as the bacterium Bacillus sphaericus isolate 2362. At 24 h after application, the fungus-induced mortality on mosquito larvae was significantly lower than the mortality caused by the bacterium. By the second day, almost no activity by the fungus was observed. Results suggest that M. anisopliae isolate 1037 has potential for use in mosquito control programmes.     

Evolutionary history of the European free-tailed bat,a tropical affinity species spanning across the Mediterranean Basin

The Mediterranean Basin is a global biodiversity hotspot, hosting a number of native species belonging to families that are found almost exclusively in tropical climates. Yet, whether or not these taxa were able to survive in the Mediterranean region during the Quaternary climatic oscillations remains unknown. Focusing on the European free-tailed bat (Tadarida teniotis), we aimed to (a) identify potential ancient populations and glacial refugia; (b) determine the post-glacial colonization routes across the Mediterranean; and (c) evaluate current population structure and demography. Mitochondrial and nuclear markers were used to understand T. teniotis evolutionary and demographic history. We show that T. teniotis is likely restricted to the Western Palearctic, with mitochondrial phylogeny suggesting a split between an Anatolian/Middle East clade and a European clade. Nuclear data pointed to three genetic populations, one of which is an isolated and highly differentiated group in the Canary Islands, another distributed across Iberia, Morocco, and France, and a third stretching from Italy to the east, with admixture following a pattern of isolation by distance. Evolutionary and demographic reconstruction supports a pre-Last Glacial Maximum (LGM) colonization of Italy and the Anatolian/Middle East, while the remaining populations were colonized from Italy after the Younger Dryas. We also found support for demographic expansion following the Iberian colonization. The results show that during the LGM T. teniotis persisted in Mediterranean refugia and has subsequently expanded to its current circum-Mediterranean range. Our findings raise questions regarding the physiological and ecological traits that enabled species with tropical affinities to survive in colder climates.     

Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil

Candida species are an important cause of bloodstream infections (BSI). To evaluate the epidemiological, clinical and microbiological aspects of two cohorts {1994-1999 [period 1 (P1) ]; 2000-2004 [period 2 (P2) ]} of candidaemic patients, we performed a retrospective analysis from a laboratory-based survey. A total of 388 candidaemias were identified, with an incidence of 0.20/1,000 patient-days and a significant increase in P2 vs. P1 (0.25 vs. 0.15, p = 0.04). Cancer and prior antibiotic use were frequent and Candida albicans was the most prevalent species found (42.4%). Resistance to fluconazole was found in 2.47% of the strains. No differences were observed in the species distribution of Candida during the study periods. In the P2 cohort, there were higher prevalence of elderly individuals, cardiac, pulmonary and liver diseases, renal failure, central venous catheters and antibiotic therapy. In P1, there were higher prevalence of neurological diseases and chemotherapy. The crude mortality was 55.4%. In conclusion, our incidence rates remained high. Furthermore, the distribution pattern of Candida species and the fluconazole resistance profile remained unchanged. Moreover, we found a clear trend of higher prevalence of candidaemia among the elderly and among patients with comorbidities. Finally, it is necessary to discuss strategies for the prevention and control of Candida BSI in Brazil.     

Factors associated with Diaphorina citri immigration into commercial citrus orchards in São Paulo State,Brazil

Seasonality often influences multiple aspects of vector-borne plant diseases. With respect to the management of plant disease vectors, an understanding of the factors driving seasonal changes in vector flight activity may improve management outcomes by facilitating more proactive application of control measures. One of the most challenging issues for citrus Huanglongbing management is to forecast when disease primary spread by bacteriliferous Diaphorina citri will occur. We monitored the temporal patterns of immigrating psyllids, using upwards of 1,200 traps spread among 9 citrus farms and checked on a weekly basis over 4 years. This dataset was analysed with a set of hierarchical models to estimate the effect of climatic variables on citrus foliage production (i.e. flush), and effects of climatic variables and citrus foliage dynamics on D. citri catches over the season. The results showed substantial seasonal variability in immigrating D. citri abundance, with the critical dispersal/migration period occurring between end of winter and spring. During this period, 65% of the total psyllids were collected on sticky traps. Seasonality of immigrating D. citri coincided with changes in certain climate variables, with negative effects of humidity and daily maximum temperatures, and a positive effect of rainfall amount in prior weeks. Maximum temperature and both daily minimum temperature and rainfall during prior weeks were also associated with new citrus flush production, which itself was positively related to immigrating D. citri abundance. Based on these results, citrus growers should be aware and intensify the frequency of psyllid control tactics (i.e. insecticide and/or kaolin sprays) during this period in order to prevent Ca. L. asiaticus transmission. These results are an important step towards developing the predictive framework needed to refine D. citri and huanglongbing management.     

Caspase-8 activity prevents type 2 cytokine responses and is required for protective T cell-mediated immunity against Trypanosoma cruzi infection

During Trypanosoma cruzi infection, T cells up-regulate caspase-8 activity. To assess the role of caspase-8 in T cell-mediated immunity, we investigated the effects of caspase-8 inhibition on T cells in viral FLIP (v-FLIP) transgenic mice. Compared with wild-type controls, increased parasitemia was observed in v-FLIP mice infected with T. cruzi. There was a profound decrease in expansion of both CD4 and CD8 T cell subsets in the spleens of infected v-FLIP mice. We did not find differences in activation ratios of T cells from transgenic or wild-type infected mice. However, the numbers of memory/activated CD4 and CD8 T cells were markedly reduced in v-FLIP mice, possibly due to defective survival. We also found decreased production of IL-2 and increased secretion of type 2 cytokines, IL-4 and IL-10, which could enhance susceptibility to infection. Similar, but less pronounced, alterations were observed in mice treated with the caspase-8 inhibitor, zIETD. Furthermore, blockade of caspase-8 by zIETD in vitro mimicked the effects observed on T. cruzi infection in vivo, affecting the generation of activated/memory T cells and T cell cytokine production. Caspase-8 is also required for NF-kappaB signaling upon T cell activation. Blockade of caspase-8 by either v-FLIP expression or treatment with zIETD peptide decreased NF-kappaB responses to TCR:CD3 engagement in T cell cultures. These results suggest a critical role for caspase-8 in the establishment of T cell memory, cell signaling, and regulation of cytokine responses during protozoan infection.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.     

Presence of fimH,mrkD, and irp2 Virulence Genes in KPC-2-Producing Klebsiella pneumoniae Isolates in Recife-PE,Brazil

Klebsiella pneumoniae strains can produce different virulence factors, such as fimbrial adhesins and siderophores, which are important in the colonization and development of the infection. The aims of this study were to determine the occurrence of fimH, mrkD, and irp2 virulence genes in 22 KPC-2-producing K. pneumoniae isolates as well as 22 not producing-KPC isolates, from patients from different hospitals in Recife-PE, Brazil, and also to analyze the clonal relationship of the isolates by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The genes were detected by PCR and DNA sequencing. The bla _KPC-2 gene was identified in 22 KPC-positive isolates. On analyzing the antimicrobial susceptibility profile of the isolates, it was detected that polymyxin and amikacin were the antimicrobials of best activity against K. pneumoniae. On the other hand, five isolates exhibited resistance to polymyxin. In the KPC-positive group, was observed a high rate of resistance to cephalosporins, followed by carbapenems. Molecular typing by ERIC-PCR detected 38 genetic profiles, demonstrating a multiclonal spread of the isolates analyzed. It was observed that the virulence genes irp2, mrkD, and fimH were seen to have together a higher frequency in the KPC-positive group. The accumulation of virulence genes of KPC-positive K. pneumoniae isolates, observed in this study, along with the multi-resistance impose significant therapeutic limitations on the treatment of infections caused by K. pneumoniae.