Intravesicular Factors Controlling Exocytosis in Chromaffin Cells

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many secretory cell types including neurons. LDCV accumulate solutes at high concentrations (catecholamines, 0.5–1 M; ATP, 120–300 mM; or Ca²⁺, 40 mM (Bulenda and Gratzl Biochemistry 24:7760–7765, 1985). Solutes seem to aggregate to a condensed matrix to elude osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delayed release of catecholamines during exocytosis. The aggregation of solutes occurs due to a specific H⁺ pump denominated V-ATPase that maintains an inner acidic media (pH ≈5.5). This pH gradient against cytosol is also responsible for the vesicular accumulation of amines and Ca²⁺. When this gradient is reduced by modulation of the V-ATPase activity, catecholamines and Ca²⁺ are moved toward the cytosol. In addition, some drugs largely accumulate inside LDCV and not only impair the accumulation of natural solutes, but also act as false neurotransmitters when they are co-released with catecholamines. There is much experimental evidence to conclude that the physiological modulation of vesicle pH and the manipulation of intravesicular media with drugs affect the LDCV cargo and change the kinetics of exocytosis. Here, we will present some experimental data demonstrating the participation of drugs in the kinetics of exocytosis through changes in the composition of vesicular media. We also offer a model to explain the regulation of exocytosis by the intravesicular media that conciliate the experimentally obtained data.     

Induction of phytochelatins and antioxidant defence system in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Vigna radiata</Emphasis> in response to chromium treatments

Growth performance, chromium (Cr) accumulation potential and induction of antioxidative defence system and phytochelatins (PCs) were studied in hydroponically grown Brassica juncea (Indian mustard) and Vigna radiata (mungbean) at various levels of Cr treatments (0, 50, 100, 200 μM Cr). B. juncea accumulated twofolds and threefolds higher Cr in root and shoot, respectively than in V. radiata. Compared to B. juncea, V. radiata was found to be particularly sensitive to Cr as observed by the severity and development of Cr toxicity symptoms and decreased growth. Induction of PC and enzymes of antioxidant defence system were monitored as plant’s primary and secondary metal detoxifying responses, respectively. There was induction of PC and enzymes of antioxidant defence system in both the plants. PCs were induced significantly in roots and shoot of both the plants at all the levels of Cr treatments. Significantly higher activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) were observed in shoot of B. juncea than V. radiata at all the levels of Cr treatments. Induction of PCs along with antioxidant defence system in response to Cr stress suggests the cumulative role of PCs and antioxidants in conferring tolerance against accumulated Cr in B. juncea, and thereby signifies the suitability of this plant as one of the potential remediators of Cr.     

Dispersal mechanisms of the narrow endemic <Emphasis Type="Italic">Polygala vayredae</Emphasis>: dispersal syndromes and spatio-temporal variations in ant dispersal assemblages

This study assesses the dispersal mechanisms of the narrow endemic Polygala vayredae, analysing the functioning of its dispersal syndromes (anemochory and myrmecochory), the spatio-temporal variability of the disperser assemblage, foraging behaviour and dispersal ability, and the role of the elaiosome in ant attraction and seed germination. The dispersion of diaspores begins when either (1) capsules or seeds fall beneath the mother plant (barochory) or (2) the seeds are directly collected in the suspended capsules by ants (myrmecochory). As capsules frequently open and expose/disseminate seeds before leaving the mother plant, the adaptation for anemochory appears to be reduced and rarely functional, possibly with only occasional events of long-distance dispersal (e.g. under extreme weather conditions). P. vayredae is essentially myrmecochorous and a diverse array of ant species are involved in seed manipulation, with the elaiosome playing a major role in ant attraction. From the plant’s perspective for dispersal, the majority of ant species had a positive interaction with the seeds, but negative and potential neutral interactions were also observed. Overall, dispersal distances were limited and were mainly determined by ant body size. The frequency of interactions and the ant assemblage varied significantly both spatially and temporally, and these factors may have an effect on directing or disrupting the selection of plant traits. Low seed predation and similar germination rates of intact seeds compared with seeds without elaiosome indicate that seed predator avoidance and seed germination improvement after ant manipulation are not among the selective advantages of myrmecochory operating at present. Dispersal mechanisms that enhance seed dispersal within the population and only occasionally lead to long-distance dispersal events, along with the rarity and patchiness of suitable habitats, may be the main factors explaining the actual density and narrow distribution of this species.     

Insulin Mimetic Effect of Tungsten Compounds on Isolated Rat Adipocytes

Investigations of effective, orally active, and safe antidiabetic metallopharmaceuticals have been carried out during the last two decades. It has been reported that tungsten compounds mimic the action of insulin in intact cell systems. As insulin mimetics, the most investigated tungsten compound was sodium tungstate (ST), rarely investigated was tungstophosphoric acid (WPA), but never alanine complex of tungstophosphoric acid (WPA-A). In this study, the insulin mimetic activity of three different tungsten compounds, ST, WPA, and WPA-A, was evaluated by means of in vitro measurements of the glucose uptake and inhibition of free fatty acids release from epinephrine-treated isolated rat white adipocytes. We investigated the influence of concentration (lower and higher, 0.1 and 1.0 mM, respectively) and solvent: isotonic salt solution—saline (0.9% w/v of NaCl) and dimethyl sulfoxide (DMSO; 2% v/v), on the biological effect of tested compounds. Our experimental data showed that all of the three investigated tungsten compounds possess insulin mimetic activity in vitro on the isolated adipocytes. Influence of concentration and solvents on insulin mimetic effect for the certain tungsten compounds were: WPA was shown effect independently of concentration and solvents; higher concentration and DMSO were significant decreasing insulin mimetic effect of ST; lower concentration and saline led to decreasing effect of WPA-A. Generally, there were no differences in insulin mimetic effect of three tungsten compounds in lower concentration and dissolved in DMSO. When saline was used as solvent, it was needed higher concentration of investigated compounds to accomplish the same effect. In conclusion, our results suggest that low concentration (0.1 mM) of ST, WPA, and WPA-A dissolved in 2% DMSO could be the good candidates for in vivo investigation of their antidiabetic properties.     

Noninvasive genetic studies of brown bears using power poles

One difficulty in the conservation of endangered wildlife is the lack of reliable information on its status. This lack of knowledge can often be attributed to financial and logistic constraints as well as the lack of trained personnel to collect data. We test a simple method to study bears in the southern Balkans by inspecting power poles, which are used by bears for marking and rubbing purposes. We created a network of barbed-wire fitted poles for the collection of hair samples, evenly distributed throughout six study areas. During 87 sampling sessions in the main study area, we collected 191 samples and identified six microsatellite loci that were variable enough for individual bear identification. The most and best-quality hair samples were collected during the mating period, and DNA was most successfully extracted from samples remaining <4 weeks in the field. In the six study areas, we identified 47 bears. An advantage of using power poles for hair sampling is their availability and accessibility; no bait is required, and the network can be easily set up. A drawback may be an unequal capture probability of sex and age classes of bears. Despite this limitation, using power poles proved to be a simple and cheap method for the noninvasive genetic study of bears that did not require any prior knowledge on habitat use and activity patterns. The method is suitable for large-scale surveys to estimate distribution and relative densities of bears and could also be applied for studying other species.     

An Examination of the Differential Sensitivity to Ketolide Antibiotics in <Emphasis Type="Italic">ermB</Emphasis> Strains of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC₅₀ values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC₅₀ values for inhibition of translation and subunit formation in S. pneumoniae (ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC₅₀ values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.     

Use of low nutrient enrichments to access novel amylase genes in silent diversity of thermophiles

By combining low nutrient enrichments and molecular methods, a high diversity of new amylase genes was detected in a neutral sulphide-rich hot spring in Iceland. Enrichments based on hot spring water and low concentrations of starch were used to select slow-growing, starch-degrading microorganisms. Six enrichments had in total 17 bacterial types detected by 16S rRNA analysis, mostly related to the Thermus-Deinococcus group, green non-sulphur bacteria, gram positives, and uncultivated new candidate divisions. No Archaea were found. The apparent 16S rRNA species composition of the enrichments was very different from that of the microbial mat in the same hot spring. DNA samples obtained from 4 enrichments and from hot spring biomass were screened by PCR for amylase genes in glycoside-hydrolase family 13. Degenerate primers, based on conserved amino acid sequences from multiplealignments of family 13, enabled the detection of 18 amylase sequence types in the enrichments, including -amylases, -glucosidases, 1,4--glucan branching enzymes, cyclomaltodextrin hydrolases, maltogenic amylases and neopullulanases, and unspecified family 13 glycoside-hydrolases. Only one unique neopullulanase sequence, also found in most of the enrichments, was detected in the hot spring biomass DNA. The results suggest that the enrichment method combined with sequence-based screening is an efficient way to access the silent, i.e. not detectable, gene diversity in natural environments.     

Biological treatment of shrimp production wastewater

Over the last few decades, there has been an increase in consumer demand for shrimp, which has resulted in its worldwide aquaculture production. In the United States, the stringent enforcement of environmental regulations encourages shrimp farmers to develop new technologies, such as recirculating raceway systems. This is a zero-water exchange system capable of producing high-density shrimp yields. The system also produces wastewater characterized by high levels of ammonia, nitrate, nitrite, and organic carbon, which make waste management costs prohibitive. Shrimp farmers have a great need for a waste management method that is effective and economical. One such method is the sequencing batch reactor (SBR). A SBR is a variation of the activated sludge biological treatment process. This process uses multiple steps in the same reactor to take the place of multiple reactors in a conventional treatment system. The SBR accomplishes equalization, aeration, and clarification in a timed sequence in a single reactor system. This is achieved through reactor operation in sequences, which includes fill, react, settle, decant, and idle. A laboratory scale SBR was successfully operated using shrimp aquaculture wastewater. The wastewater contained high concentrations of carbon and nitrogen. By operating the reactors sequentially, namely, aerobic and anoxic modes, nitrification and denitrification were achieved as well as removal of carbon. Ammonia in the waste was nitrified within 4 days. The denitrification of nitrate was achieved by the anoxic process, and 100% removal of nitrate was observed within 15 days of reactor operation.     

Increased constraints on MC4R during primate and human evolution

The melanocortin 4 receptor (MC4R) is routinely investigated for the role it plays in human obesity, as mutations in MC4R are the most common dominantly inherited form of the disease. As little is known about the evolutionary history of this locus, we investigated patterns of variation at MC4R in a worldwide sample of 1,015 humans from 51 populations, and in 8 central chimpanzees. There is a significant paucity of diversity at MC4R in humans, but not in chimpanzees. The spectrum of mutations in humans, combined with the overall low level of diversity, suggests that most (if not all) of the observed non-synonymous polymorphisms are likely to be transient deleterious mutations. The MC4R coding region was resequenced in 12 primate species and sequences from an additional 29 vertebrates were included in molecular evolutionary analyses. MC4R is highly conserved throughout vertebrate evolution, and has apparently been subject to high levels of continuous purifying selection that increased approximately threefold during primate evolution. Furthermore, the strong selection extends to codon usage bias, where most silent mutations are expected to be either quickly fixed or removed from the population, which may help explain the unusually low levels of silent polymorphisms in humans. Finally, there is a significant tendency for non-synonymous mutations that impact MC4R function to occur preferentially at sites that are identified by evolutionary analyses as being subject to very strong purifying selection. The information from this study should help inform future epidemiological investigations of MC4R. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.