Comparative biochemical profiling during the stages of acquisition and development of somatic embryogenesis in African oil palm (Elaeis guineensis Jacq.)

In this study, the different stages of somatic embryogenesis (SE) of the African oil palm (Elaeis guineensis Jacq.) were characterized biochemically. The total soluble sugars, starch, total free amino acids, and total proteins were extracted, identified and quantified at various stages of embryogenesis: zygotic embryos (initial explants), primary calluses, embryogenic calluses, calluses with pro-embryos, globular embryos, differentiated somatic embryos, and regenerated plants. It was found that at the onset of induction of SE, the level of soluble sugars in the tissues of the explants fell by half. During this period, the total soluble sugars present in the cultures consisted basically of glucose and fructose. In the process of regeneration and maturation, the concentrations of soluble sugars gradually increased, reaching the highest values in the last two stages of development. At this stage, the disaccharide sucrose accounts for more than 80 % of the composition of total soluble sugars in the explants. Compared to starch, we found that the concentrations thereof in developing tissues are inversely proportional to that of soluble sugars virtually throughout embryogenic development. As for free amino acids, we found that after 30 days of induction until formation of the embryogenic calluses, there is an accentuated synthesis of total free amino acids in the explant tissue. In this stage, there was a significant increase in the levels of alanine and serine in the tissues. However, after the formation of the embryogenic calluses, the levels of total free amino acids present in the cultures become stabile and remain constant until the end of cultivation. Similar results were found for total protein, which also showed a significant increase at the onset of induction, undergoing slight changes during the remainder of the cultivation.     

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.     

Semi-automated recognition of protozoa by image analysis

A programme was created to semi-automatically analyse protozoal digitised images. Principal Component Analysis technique was used for species identification. After data collection and mathematical treatment, a three-dimensional representation was generated and several protozoa (Opercularia, Colpidium, Tetrahymena, Prorodon, Glaucoma and Trachelophyllum) species could be positively identified.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Aporphine Alkaloids from Ocotea puberula with Anti-Trypanosoma Cruzi Potential – Activity of Dicentrine-β-N-Oxide in the Plasma Membrane Electric Potentials

One new aporphine, dicentrine-β-N-oxide ( 1 ), together with five related known alkaloids dehydrodicentrine ( 2 ), predicentrine ( 3 ), N-methyllaurotetanine ( 4 ), cassythicine ( 5 ), and dicentrine ( 6 ) were isolated from the leaves of Ocotea puberula (Lauraceae). Antiprotozoal activity of the isolated compounds was evaluated in vitro against trypomastigote forms of Trypanosoma cruzi. Among the tested compounds, alkaloid 1 exhibited higher potential with EC₅₀ value of 18.2 μM and reduced toxicity against NCTC cells (CC₅₀>200 μM – SI>11.0), similar to positive control benznidazole (EC₅₀ of 17.7 μM and SI=10.7). Considering the promising results of dicentrine-β-N-oxide ( 1 ) against trypomastigotes, the mechanism of parasite death caused by this alkaloid was investigated. As observed, this compound reached the plasma membrane electric potential directly after 2 h of incubation and triggered mitochondrial depolarization, which probably leads to trypomastigote death. Therefore, dicentrine-β-N-oxide ( 1 ), reported for the first time in this work, can contribute to future works for the development of new trypanocidal agents.     

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease

Background

Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations.

Methods and Findings

Our data established that CH displayed increased expression of CD32⁺ and CD56⁺ in monocytes and enhanced frequency of NK Granzyme A⁺ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8⁺ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8⁺ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8⁺ T-cells, as the most reliable biomarkers of potential use for clinical applications.

Conclusions

Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.     

Reproductive biology and pollination of southeastern Brazilian Stanhopea Frost ex Hook. (Orchidaceae)

Reproductive biology and pollination of Stanhopea lietzei and Stanhopea insignis were studied in a semi-deciduous mesophytic forest in the Serra do Japi (SJ), and in the coastal plain of Picinguaba, both in the State of São Paulo, Brazil. Floral morphology, pollination, breeding system and fruit set of both species were investigated. S. lietzei and S. insignis are pollinator-specific, being pollinated by male bees of Eufriesea (Apidae, Euglossini), which collect the fragrance produced by pluricellular osmophores at the base of the saccate hypochile. S. lietzei and S. insignis were pollinated by Eufriesea pulchra and Eufriesea purpurata, respectively. Observations using substances present in the floral fragrance of both studied species as chemical baits were also performed. E. purpurata was attracted by benzyl alcohol, the major compound of the perfume of S. insignis, while E. pulchra was attracted by none of the compounds used. Both studied Stanhopea are self-compatible but pollinator dependent. Self-pollination, however, tends to be avoided by floral mechanisms. In experimental self- and cross-pollinations the proportion of fruit abortion was high and related to resource limitation. The reproductive success of S. lietzei and S. insignis was low as a consequence of deficient pollen transference while pollinator scarcity was the main factor.     

Impaired cytoprotective function of muscle in human gallbladders with cholesterol stones

Acute cholecystitis develops in gallbladders (GB) with excessive bile cholesterol (Ch). Increased membrane Ch content affects membrane function and may affect PGE(2) receptors involved in the cytoprotection against acute inflammation. This study was aimed at determining whether the cytoprotective response to PGE(2) is affected by lithogenic bile with Ch. Muscle cells from human GB with cholesterol stones (ChS) or pigment stones (PS) were obtained by enzymatic digestion. PGE(2) levels were measured by radioimmunoassay, and activities of superoxide dismutase (SOD) and catalase were assayed by spectrophotometry. The contraction in response to H(2)O(2) in muscle cells from PS was 14 +/- 0.3%, not different from normal controls, and decreased after the cells were incubated with Ch-rich liposomes (P < 0.05), which increase the Ch content in the plasma membranes. In muscle cells from GB with ChS, H(2)O(2)-induced contraction was only 9.2 +/- 1.3% and increased to 14 +/- 0.2% after Ch-free liposome treatment to remove Ch from the plasma membranes (P < 0.01). H(2)O(2) caused a similar increase in the levels of lipid peroxidation and PGE(2) content in muscle cells from GBs with ChS and PS. However, the activities of SOD and catalase were significantly lower in muscle cells from GBs with ChS compared with those with PS. The binding capacity of PGE(2) receptors was also significantly lower in muscle cells from GBs with ChS compared with those with PS. In conclusion, the cytoprotective response to reactive oxygen species is reduced in muscle cells from GBs with ChS despite a normal increase in the cellular levels of PGE(2). This impaired cytoprotective response may be due to a dysfunction of PGE(2) receptors with decreased binding capacity resulting from excessive Ch levels in the plasma membrane.     

Drosophila wing-spot test for genotoxic assessment of pollutants in water samples from urban and industrial origin

The Caí River (Rio Grande do Sul, Brazil) is an important watercourse that receives large amounts of industrial and untreated municipal discharges in its lower course. We employed the SMART in Drosophila melanogaster to evaluate the genotoxicity of surface waters collected from Caí sites receiving direct sewage discharge: from Montenegro (Km 52) and from S?o Sebasti?o do Caí (Km 78 and 80), and from two sites under the industrial influence (Km 13.6 and 18.6). The genotoxic analysis included three collections: March, June and September 1999, which were tested at crude sample and at 50 and 25% concentrations. Considering the industrial samples from Km 18.6 and 13.6, collected in March, June and September 1999, they were characterized as not having genetic toxicity. The urban samples collected in March--Km 52, 78 and 80--showed a significant increment in the frequencies of total spots. In Km 52 and 78 the genotoxic effect was associated to both mutational and recombinational events, although for Km 80 the increases observed were mainly related to the occurrence of homologous recombination. Moreover, the Km 80 crude sample from June and all the concentrations analyzed for Km 52 in September were also able to induce mitotic recombination. These effects were only observed in the ST cross, demonstrating the genotoxins present in the urban discharges act by direct interaction with the DNA of the somatic cells. The SMART in D. melanogaster was shown to be highly sensitive to detect genotoxic agents present in the aquatic environment, and must be better exploited for monitoring areas under anthropogenic discharges.     

Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca

The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species.