PACAP Enhances Axon Outgrowth in Cultured Hippocampal Neurons to a Comparable Extent as BDNF

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activities including modulation of synaptic plasticity and memory, hippocampal neurogenesis, and neuroprotection, most of which are shared with brain-derived neurotrophic factor (BDNF). Therefore, the aim of this study was to compare morphological effects of PACAP and BDNF on primary cultured hippocampal neurons. At days in vitro (DIV) 3, PACAP increased neurite length and number to similar levels by BDNF, but vasoactive intestinal polypeptide showed much lower effects. In addition, PACAP increased axon, but not dendrite, length, and soma size at DIV 3 similarly to BDNF. The PACAP antagonist PACAP6–38 completely blocked the PACAP-induced increase in axon, but not dendrite, length. Interestingly, the BDNF-induced increase in axon length was also inhibited by PACAP6–38, suggesting a mechanism involving PACAP signaling. K252a, a TrkB receptor inhibitor, inhibited axon outgrowth induced by PACAP and BDNF without affecting dendrite length. These results indicate that in primary cultured hippocampal neurons, PACAP shows morphological actions via its cognate receptor PAC₁, stimulating neurite length and number, and soma size to a comparable extent as BDNF, and that the increase in total neurite length is ascribed to axon outgrowth.     

Hydrology and water balance of Lake Peipsi

Lake Peipsi is a large (3558 km²) but shallow (up to 15.3 m deep) tripartite waterbody hydrologically investigated already since the 19th century. Surface discharge by rivers accounts for more than 80% of its water balance. The residental time of water is about two years in the whole lake but several times less in its shallower southern parts receiving the biggest rivers. The annual water regime is characterized by the highest water in spring, the average amplitude of yearly level fluctuations being 1.15 m. There are known long-term hydrological cycles of 80–90, about 22, 9–11, and even fewer years. Several temporary wind-dependent circular currents exist in the subsurface layers. Alternating transitional currents occur in the narrowest part of the lake. Five different periods are distinguishable in the annual thermic cycle. The duration of the stable ice cover is up to five months (December-April) in the shallower parts but a shorter time in the centre of the lake. The maximum surface temperature in July usually reaches 21–22°C in the open regions but considerably higher (up to 27–28°C in some years) on shallows. The unstable summer stratification is often disturbed by waves and currents. Biological summer, with surface temperatures over 10°C, lasts on an average 134 days.     

Isolated and repeated stroke-like episodes in a middle-aged man with a mitochondrial ND3 T10158C mutation: a case report

Background

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, is the most common phenotype of mitochondrial disease. It often develops in childhood or adolescence, usually before the age of 40, in a maternally-inherited manner. Mutations in mitochondrial DNA (mtDNA) are frequently responsible for MELAS.

Case presentation

A 55-year-old man, who had no family or past history of mitochondrial disorders, suddenly developed bilateral visual field constriction and repeated stroke-like episodes. He ultimately presented with cortical blindness, recurrent epilepsy and severe cognitive impairment approximately 6 months after the first episode. Genetic analysis of biopsied biceps brachii muscle, but not of peripheral white blood cells, revealed a T10158C mutation in the mtDNA-encoded gene of NADH dehydrogenase subunit 3 (ND3), which has previously been thought to be associated with severe or fatal mitochondrial disorders that develop during the neonatal period or in infancy.

Conclusion

A T10158C mutation in the ND3 gene can cause atypical adult-onset stroke-like episodes in a sporadic manner.

     

Helix 8 of the leukotriene B4 receptor is required for the conformational change to the low affinity state after G-protein activation

Recent studies have revealed that G-protein-coupled receptors contain a putative cytoplasmic helical domain, helix 8. Leukotriene B4 (LTB4) receptor 1 derivatives with truncated or mutated helix 8 showed much higher LTB4 binding than wild-type (WT) receptors. Similar to the WT receptor, LTB4 promoted guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in these mutants. Unlike the WT receptor, however, the addition of GTPgammaS did not inhibit LTB4 binding to the mutant receptors. Scatchard analyses revealed that mutants maintained high affinity for LTB4, even in the presence of excess GTPgammaS. Consistently, mutant receptors showed a more prolonged Ca2+ mobilization and cellular metabolic activation than the WT receptor. From mutational studies and three-dimensional modeling based on the structure of bovine rhodopsin, we conclude that the helix 8 of LTB4 receptor 1 plays an important role in the conformational change of the receptor to the low affinity state after G-protein activation, possibly by sensing the status of coupling Galpha subunits as GTP-bound.     

Accounting for tree spatial distribution in a comparison of plot sizes and shapes in dense forest and woodland in Benin (West Africa)

The study examined simultaneously, the effect of tree spatial distribution, inventory plot size and shape on the estimation error of basal area in two contrasting environments. Twenty and fifteen square plots of 1 ha each (divided into 100 quadrats of 0.01 ha) were randomly set in dense forest and woodland, respectively. Thirteen subplots of various shapes and sizes were obtained from the association of adjacent quadrats. Estimation error was calculated using residual mean square of one‐way ANOVA, based on replications of subplot within 1 ha plots. Tree spatial distribution was measured using Green index. Weighted linear regression and mixed effect models were applied to Box & Cox transformed data. In general, the estimation error of basal area decreased with increase in subplot size. However, the effects of tree spatial distribution and plot shape varied with the vegetation type. Where trees tended to be aggregated, estimation error increased with degree of aggregation, and rectangular plots of 0.24 ha produced an acceptable precision. It was concluded that 0.24 ha rectangular plots can be used in tropical environments where the target parameters vary constantly according to one direction, while square plots of the same size are optimal for reliable analysis in case of randomness.     

The Rice Aquaporin Lsi1 Mediates Uptake of Methylated Arsenic Species

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher V_max than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.Arsenic (As) contamination affects millions of people worldwide, particularly in South Asia where As-contaminated groundwater has been extracted for drinking (Chakraborti et al., 2002; Nordstrom, 2002). Recent studies have shown that foods, especially rice (Oryza sativa), are an important source of inorganic As to populations dependent on a rice diet (Kile et al., 2007; Ohno et al., 2007; Mondal and Polya, 2008). Paddy rice is more efficient than other cereal crops in accumulating As (Williams et al., 2007). This is because anaerobic conditions in submerged paddy soils lead to mobilization of arsenite [As(III); Takahashi et al., 2004; Xu et al., 2008], which is then taken up by rice roots mainly through the highly efficient transport pathway for silicon (Si; Ma et al., 2008). The relatively high accumulation of As in rice is of concern, as it may pose a significant health risk (Zhu et al., 2008; Meharg et al., 2009).A number of As species may be present in soil depending on soil conditions and the history of As contamination. These include arsenate [As(V)], As(III), and methylated As species such as monomethylarsonic acid [MMA(V): CH₃AsO(OH)₂] and dimethylarsinic acid [DMA(V): (CH₃)₂AsO(OH)]. As(V) is the main species in aerobic soils, while As(III) dominates in anaerobic environments such as flooded paddy soils. Both MMA(V) and DMA(V) have been found in paddy soils (Takamatsu et al., 1982), which may have been derived from microbial and algal biomethylation and/or past uses of methylated As compounds. MMA(V), as sodium or calcium salt, and DMA(V), as sodium salt or free acid (also called cacodylic acid), are herbicides widely used for weed control on cotton (Gossypium hirsutum), orchards, and lawns, or as a defoliant of cotton (U.S. Environmental Protection Agency, 2006). Conversion of cotton fields for the production of paddy rice in the United States may be a reason for the high levels of methylated As reported for the U.S. rice (Meharg et al., 2009).The mechanism of As(V) uptake by plants through the phosphate transport system has been well established (for review, see Zhao et al., 2009). In contrast, As(III) is taken up into the cells by aquaglyceroporins in Escherichia coli, yeast (Saccharomyces cerevisiae), and mammalian tissues (for review, see Bhattacharjee and Rosen, 2007). Recent studies have shown that several plant aquaporin channels belonging to the Nodulin 26-like Intrinsic Protein (NIP) subfamily are permeable to As(III) when expressed heterologously in yeast (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008). The rice Si transporter Lsi1 (OsNIP2;1; Ma et al., 2006) is also permeable to As(III) when expressed in yeast or Xenopus laevis oocytes (Ma et al., 2008). Furthermore, the lsi1 rice mutant lost 57% of the influx capacity for As(III) compared to the wild type in short-term assays, suggesting that Lsi1 is an important entry route for As(III) (Ma et al., 2008). In rice roots, a second Si transporter, Lsi2, functions as an efflux carrier to transport Si efflux from the exodermis and endodermis cells toward the stele for xylem loading (Ma et al., 2007). This transporter also mediates As(III) efflux; two independent lsi2 mutants had 73% to 91% lower concentrations of As(III) in the xylem sap than their wild types (Ma et al., 2008). The shared uptake pathway between Si (silicic acid) and As(III) (arsenous acid) is consistent with their physiochemical properties; both are present predominantly as undissociated neutral molecules at the normal environmental and physiological pH range (pK_a = 9.2, >99% undissociated at pH ≤ 7.0), and the two molecules have similar sizes.The uptake mechanisms of methylated As species by plant roots are not known. Previous studies showed that both MMA(V) and DMA(V) can be taken up by roots and translocated to shoots in a number of plant species (Marin et al., 1992; Carbonell-Barrachina et al., 1998, 1999; Burló et al., 1999). Marin et al. (1992) found that uptake by rice followed the order of As(III) > MMA(V) > As(V) > DMA (V), although DMA(V) was more efficiently translocated from roots to shoots than other As species. Raab et al. (2007) reported large variations in the absorption and translocation efficiencies for As(V), MMA(V), and DMA(V) among the 46 plant species tested. On average, root absorption of As(V) was 2.5- and 5-times higher than MMA(V) and DMA(V), respectively. The translocation efficiency, defined as the shoot-to-root concentration ratio after 24-h exposure, was highest for DMA(V) (0.8), followed by MMA(V) (0.3) and As(V) (0.09). The concentration-dependent uptake kinetics of MMA(V) in rice roots could be described by the Michaelis-Menten equation, whereas the limited uptake of DMA(V) appeared to be linear in relation to the increasing concentration in the uptake medium (Abedin et al., 2002). Abbas and Meharg (2008) showed that DMA(V) uptake by maize (Zea mays) seedlings was enhanced by more than 10-fold by a pretreatment of phosphorus starvation; this compared with only 2-fold increase in As(V) uptake. They thought that DMA(V) might be taken up by the phosphate transporters, or that phosphorus starvation altered expression of a range of membrane transporters or even membrane permeability itself.In addition to the root uptake of methylated As species, some plants appear to be able to biomethylate As, but the pathway and enzymology remains unclear (Wu et al., 2002; Zhao et al., 2009). In microbes, As methylation follows the Challenger pathway involving repeated steps of As reduction and oxidative methylation (Bentley and Chasteen, 2002). As(V) is first reduced to As(III), which is methylated by S-adenosylmethyltransferase using S-adenosyl-l-Met as the methyl donor. The product of this reaction is pentavalent MMA(V), which is reduced by a reductase to trivalent MMA(III) with thiols (e.g. glutathione). Methylation and reduction steps continue to produce di- and trimethyl As compounds. MMA(III) and DMA(III) are intermediates in the As methylation pathway, which is not very stable (Gong et al., 2001). In rice grain, DMA(V) is the main form of methylated As, and can account for up to 80% of the total As (Zavala et al., 2008; Meharg et al., 2009). In light of the significant presence of methylated As in rice, it is important to elucidate the transport and assimilation pathways of these As species in plants.In this study, we present evidence that MMA(V) and DMA(V) are taken up by rice roots, at least partly, through the NIP aquaporin channel Lsi1, and that this process is strongly pH dependent. We also show that MMA(V) can be reduced to MMA(III) in planta.     

Mechanism of modulation of [H]raclopride binding to dopaminergic receptors in rat striatal membranes by sodium ions

The mechanism of modulation of [³H]raclopride binding to dopaminergic receptors in rat brain striatal membranes by sodium ions was studied by means of equilibrium and kinetic measurements. Among different mono- and divalent cations studied, only sodium and lithium ions significantly enhanced [³H]raclopride binding to rat striatal membranes, but the effect of lithium was considerably smaller if compared with that of sodium. The equilibrium binding studies revealed that the increase in Na⁺ concentration from 0.5 to 150 mM increased both the radioligand affinity and the number of binding sites. The meaning of these changes was established by kinetic studies, which yielded hyperbolic plots of [³H]raclopride binding rate constants over the radioligand concentration. These plots correspond to the two-step ligand binding reaction mechanism, involving fast binding equilibrium followed by a slow isomerization of the receptor-antagonist complex. Sodium ions did not influence the antagonist affinity for the receptor sites in the first step of the binding process, nor the rate of isomerization of the receptor-ligand complex, but slowed down the rate of deisomerization. This led to a change in the value of the receptor-ligand dissociation constant K_d determined under equilibrium conditions. The same change in deisomerization rate was also sufficient to alter the receptor density (B_max), measured by the conventional ligand binding procedure.     

Substantial increase of the inhibitory activity of Mirabilis antiviral protein by an elimination of the disulfide bond with genetic engineering.

Mirabilis antiviral protein (MAP) is a rigid, heat-stable protein composed of 250 amino acids with an intramolecular disulfide bond. MAP inhibits the in vitro protein synthesis of rabbit reticulocyte with approximately one-thirtieth the activity of the ricin A chain, a homologous protein with no such bond (Habuka, N., Murakami, Y., Noma, M., Kudo, T., and Horikoshi, K. (1989) J. Biol. Chem. 264, 6629-6637; Habuka, N., Akiyama, K., Tsuge, H., Miyano, M., Matsumoto, T., and Noma, M. (1990) J. Biol. Chem. 265, 10988-10992). The bond is presumed to induce some structural perturbation that alters the mode of interaction with the substrate ribosome and thus lowers the activity. To confirm this hypothesis, a mutant MAP gene in which the codons of both cysteines were replaced by those of serines was constructed and expressed in Escherichia coli, and its product (C36/22OS) was purified. In a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, C36/220S showed the same mobility as that of MAP reduced by 2-mercaptoethanol, whereas nonreduced MAP showed faster migration. The inhibitory activity of C36/220S was approximately 22 times higher than that of native MAP, that is the mutant had an IC50 of 0.16 nM for the protein synthesis of the rabbit reticulocyte system, whereas the native MAP had an IC50 of 3.5 nM. The results indicate that the activity of MAP is increased by the elimination of the disulfide bond, and this supports the hypothesis.