Hinokinin biosynthesis in Linum corymbulosum Reichenb

Due to their peculiar stereochemistry and numerous biological activities, lignans are of widespread interest. As only a few biosynthetic steps have been clarified to date, we aimed to further resolve the molecular basis of lignan biosynthesis. To this end, we first established that the biologically active lignan (−)-hinokinin could be isolated from in vitro cultures of Linum corymbulosum. Two hypothetical pathways were outlined for the biosynthesis of (−)-hinokinin. In both pathways, (+)-pinoresinol serves as the primary substrate. In the first pathway, pinoresinol is reduced via lariciresinol to secoisolariciresinol by a pinoresinol–lariciresinol reductase, and methylenedioxy bridges are formed later. In the second pathway, pinoresinol itself is the substrate for formation of the methylenedioxy bridges, resulting in consecutive production of piperitol and sesamin. To determine which of the proposed hypothetical pathways acts in vivo , we first isolated several cDNAs encoding one pinoresinol-lariciresinol reductase ( PLR-Lc1 ), two phenylcoumaran benzylic ether reductases ( PCBER-Lc1 and PCBER-Lc2 ), and two PCBER-like proteins from a cDNA library of L. corymbulosum. PLR-Lc1 was found to be enantiospecific for the conversion of (+)-pinoresinol to (−)-secoisolariciresinol, which can be further converted to give (−)-hinokinin. Hairy root lines with significantly reduced expression levels of the plr-Lc1 gene were established using RNAi technology. Hinokinin accumulation was reduced to non-detectable levels in these lines. Our results strongly indicate that PLR-Lc1 participates in (−)-hinokinin biosynthesis in L. corymbulosum by the first of the two hypothetical pathways via (−)-secoisolariciresinol.     

Short-term variation of nutritive and metabolic parameters in <Emphasis Type="Italic">Temora longicornis</Emphasis> females (Crustacea,Copepoda) as a response to diet shift and starvation

Changes in fatty acid patterns, digestive and metabolic enzyme activities and egg production rates (EPR) were studied in the small calanoid copepod Temora longicornis. Female copepods were collected in spring 2005 off Helgoland (North Sea). In the laboratory one group of copepods was fed with the cryptophycean Rhodomonas baltica for a period of 3 days. Another group of copepods was maintained without food. According to the fatty acid patterns, animals from the field were feeding on a more detrital, animal-based and to a minor extent to a diatom-based diet. Under laboratory conditions, females rapidly accumulated fatty acids such as 18:4 (n-3), 18:3 (n-3) and 18:2 (n-6) which are specific of R. baltica. Diatom-specific fatty acids such as 16:1 (n-7) were strongly reduced. In fed animals the activities of digestive and metabolic enzymes remained constant and egg production rates were highest on day 2. Starving animals, in contrast, showed significantly reduced faecal pellet production and EPR. Proteolytic enzyme activity decreased rapidly within 24 h and remained at a low level until the end of the experiment. Citrate synthase decreased continuously as well. T. longicornis rapidly reacts to dietary changes and food depletion. It has limited energy stores and, thus, strongly depends on continuous food supply.     

Rhizome dynamics and resource storage in Phragmites australis

Seasonal changes in rhizome concentrations of total nonstructural carbohydrates (TNC), water soluble carbohydrates (WSC), and mineral nutrients (N, P and K) were monitored in two Phragmites australis stands in southern Sweden. Rhizome biomass, rhizome length per unit ground area, and specific weight (weight/ length ratio) of the rhizomes were monitored in one of the stands.Rhizome biomass decreased during spring, increased during summer and decreased during winter. However, changes in spring and summer were small (< 500 g DW m-2) compared to the mean rhizome biomass (approximately 3000 g DW m^–2). Winter losses were larger, approximately 1000 g DW m-2, and to a substantial extent involved structural biomass, indicating rhizome mortality. Seasonal changes in rhizome length per unit ground area revealed a rhizome mortality of about 30% during the winter period, and also indicated that an intensive period of formation of new rhizomes occurred in June.Rhizome concentrations of TNC and WSC decreased during the spring, when carbohydrates were translocated to support shoot growth. However, rhizome standing stock of TNC remained large (> 1000 g m^–2). Concentrations and standing stocks of mineral nutrients decreased during spring/ early summer and increased during summer/ fall. Only N, however, showed a pattern consistent with a spring depletion caused by translocation to shoots. This pattern indicates sufficient root uptake of P and K to support spring growth, and supports other evidence that N is generally the limiting mineral nutrient for Phragmites.The biomass data, as well as increased rhizome specific weight and TNC concentrations, clearly suggests that reloading of rhizomes with energy reserves starts in June, not towards the end of the growing season as has been suggested previously. This resource allocation strategy of Phragmites has consequences for vegetation management.Our data indicate that carbohydrate reserves are much larger than needed to support spring growth. We propose that large stores are needed to ensure establishment of spring shoots when deep water or stochastic environmental events, such as high rhizome mortality in winter or loss of spring shoots due to late season frost, increase the demand for reserves.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Genetic population structure as indirect measure of dispersal ability in a Lake Tanganyika cichlid

Diversification and speciation processes are influenced by intrinsic (ecological specialization, dispersal) and extrinsic (habitat structure and instability) factors, but the effect of ecological characteristics on dispersal is difficult to assess. This study uses mitochondrial control region sequences to investigate the population structure and demographic history of the endemic Lake Tanganyika cichlid Neolamprologus caudopunctatus with a preference for the rock-sand interface along two stretches of continuous, rocky shoreline, and across a sandy bay representing a potential dispersal barrier. Populations along uninterrupted habitat were not differentiated; whereas, the sandy bay separated two reciprocally monophyletic clades. The split between the two clades between 170,000 and 260,000 years BP coincides with a period of rising water level following a major lowstand, and indicates that clades remained isolated throughout subsequent lake level fluctuations. Low long-term effective population sizes were inferred from modest genetic diversity estimates, and may be due to recent population expansions starting from small population sizes 45,000–60,000 years BP. Comparisons with available data from specialized rock-dwelling species of the␣same area suggest that habitat structure and lake level fluctuations determine phylogeographic patterns on large scales, while fine-scale population structure and demography are modulated by species-specific ecologies.     

Physical,Behavioural and Genetic Predictors of Adult Hypertension: The Findings of the Kaunas Cardiovascular Risk Cohort Study

Background

The roots of adult hypertension go back to childhood. This study aimed to examine the independent effects of physical, behavioural and genetic factors identified in childhood and mid-adulthood for prediction of adult hypertension.

Methods

The study subjects were participants of the Kaunas Cardiovascular Risk Cohort study started in 1977 (n = 1082, age 12–13 years). In 2012, a total of 507 individuals (63.9% of eligible sample) participated in the 35-year follow-up survey. Health examination involved measurements of blood pressure (BP), anthropometric parameters, and interview about health behaviours. Subjects were genotyped for AGT (M235T), ACE (I/D, rs4340), ADM (rs7129220), and CACNB2 (rs12258967) genes polymorphisms. A genetic risk score was calculated as the sum of the number of risk alleles at each of four single nucleotide polymorphisms.

Results

AGT TT genotype male carriers had the highest mean values of systolic BP in childhood. In females, ADM genotype AA was associated with the highest values of systolic and diastolic BP, while CACNB2 genotype CC carriers had the highest values of diastolic BP in childhood. Systolic and diastolic BP in childhood, gain in BMI from childhood to adulthood, and risky alcohol consumption predicted hypertension in middle-aged men. In women, genetic risk score together with diastolic BP in childhood and gain in BMI were significant predictors of adult hypertension. The comparison of four nested logistic regression models showed that the prediction of hypertension improved significantly after the addition of BMI gain. Genetic risk score had a relatively weak effect on the improvement of the model performance in women.

Conclusions

BP in childhood and the gain in BMI from childhood to adulthood were significant predictors of adult hypertension in both genders. Genetic risk score in women and risky alcohol consumption in men were independently related with the risk of adult hypertension.     

Calmodulin Is a Functional Regulator of Cav1.4 L-type Ca2+ Channels

Cav1.4 channels are unique among the high voltage-activated Ca²⁺ channel family because they completely lack Ca²⁺-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca²⁺ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca²⁺-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca²⁺-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca²⁺ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca²⁺ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca²⁺-dependent inactivation, whereas it does not interfere with other calmodulin effects.Retinal photoreceptors and bipolar cells contain a highly specialized type of synapse designated ribbon synapses. Glutamate release in these synapses is controlled via graded and sustained changes in membrane potential that are maintained throughout the duration of a light stimulus (1, 2). In recent years, it became clear that Cav1.4 L-type Ca²⁺ channels are the main channel subtype converting these analog input signals into corresponding permanent glutamate release (1, 3–5). In support of this mechanism, mutations in the Cav1.4 gene have been identified in patients suffering from congenital stationary night blindness type 2 and X-linked cone rod dystrophy (6–8). Individuals displaying congenital stationary night blindness type 2 as well as mice deficient in Cav1.4 typically have abnormal electroretinograms that indicate a loss of neurotransmission from the rods to second order bipolar cells, which is attributable to a loss of Cav1.4 (3).Retinal Cav1.4 channels are set apart from other high voltage-activated (HVA)³ Ca²⁺ channels by their total lack of Ca²⁺-dependent inactivation (CDI) and their very slow voltage-dependent inactivation (VDI). Recently, we and others discovered an inhibitory domain (ICDI: inhibitor of CDI) in the C-terminal tail of the Cav1.4 channel that eliminates Ca²⁺-dependent inactivation in this channel by binding to upstream regulatory motifs (9, 10). Importantly, introducing the ICDI into the backbone of Cav1.2 or Cav1.3 almost completely abolishes the CDI of these channels. Contrasting with the clear cut function, the underlying mechanism by which ICDI abolishes CDI remains controversial. It was suggested that ICDI displaces the Ca²⁺ sensor calmodulin (CaM) from binding to the proximal C terminus (10), suggesting that the binding sites of CaM and ICDI are largely overlapping or allosterically coupled to each other. Alternatively, our own data rather suggested that CaM and the ICDI domain bind to different portions of the proximal C terminus (9). We proposed that the interaction between the ICDI domain and the EF-hand, a motif with a central role for transducing CDI (11–16), switches off CDI without impairing binding of CaM to the channel. In this study, we designed experiments to differentiate between these two models. Here, using FRET in HEK293 cells, we provide evidence that in living cells, CaM is bound to the full-length C terminus of Cav1.4 (i.e. in the presence of ICDI). Furthermore, our data suggest that the steric orientation of the CaM/Cav channel complex differs between Cav1.2 and Cav1.4 channels. We show that CaM preassociation with Cav1.4 controls current density and also affects VDI. Thus, although CaM does not trigger CDI in Cav1.4 as it does in other HVA Ca²⁺ channels, it is still an important regulator of this channel.