ENZYMATIC BIOSYNTHESIS OF ETHANOLAMINE- AND CHOLINE-PHOSPHOGLYCERIDES IN RETINA DURING DEVELOPMENT. EFFECT OF LIGHT STIMULATION

Choline- and ethanolamine-phosphoglycerides (CPG and EPG) are the most abundant phospholipids of retinal membranes. We have investigated some regulatory mechanisms involved in the final steps of their biosynthesis, namely those catalysed by CDP-choline 1,2 diradyl-sn-glycerol choline phosphotransferase (CPT) and CDP-ethanolamine 1,2 diradyl-sn-glycerol ethanolamine phosphotransferase (EPT). We have studied both enzymes in the retina which offers an excellent model for the investigation of the molecular basis of the effect of its physiological stimulus, the light. In chick retina. the specific activity (SA) of EPT reached a maximum at the 18th day of embryonic life and decreased thereafter. In the case of CPT, a similar peak of SA was observed at hatching. The time of maximum SA of EPT and CPT corresponded to the period during which retinal rod outer segments are formed. The apparent K_m values of EPT and CPT determined with whole retinal homogenates for CDP-bases showed different profiles. The apparent K_m of EPT decreased during embryonic life and increased thereafter whereas the apparent K_m of CPT did not change during ontogenesis. Light stimulation of calf retinal homogenates had different effects on phosphotransferase activities. In the presence of only endogenous diacylglycerol (DAG) the SA of CPT was 2-fold higher for dark-adapted retinas, whereas no differences in EPT activities were observed. After addition of exogenous DAG (4mM) to the incubation medium, light stimulation of the retina led to a 50% increase of EPT activity whereas no effect was observed for CPT. These different effects could be related to the cyclic nucleotides present in retina before and after light stimulation. In addition all the data presented in this study indicate that, as in brain, CPT and EPT in retina are two different enzymes.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Studies on Possible Mechanisms of Early Functional Compensatory Adaptation in the Remaining Kidney

Two to 4 hours after unilateral renal exclusion in rats, urine flow rate from the remaining kidney had increased to twice the control level, whereas the filtration rate remained unchanged. After contralateral nephrectomy, NGFR was similar to that of controls, but fractional water reabsorption along proximal tubules decreased. Protein concentration in efferent arteriolar plasma, and hydrostatic pressure gradient between proximal tubules and peritubular capillaries were similar in experimental and control kidneys. Unilateral renal exclusion was followed by a rapid increase of blood pressure. Prevention of this rise depressed but did not abolish functional compensatory adaptation. The occurrence of compensatory adaptation was not affected by decreased renal perfusion pressure.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Sequence analysis of the grey mouse lemur (<Emphasis Type="Italic">Microcebus murinus</Emphasis>) <Emphasis Type="Italic">MHC class II DQ</Emphasis> and <Emphasis Type="Italic">DR</Emphasis> region

We here report the genomic organisation of the grey mouse lemur (Microcebus murinus) MHC class II DQ and DR region based on BAC clone analysis. The sequenced Mimu-MHC haplotype spans 343 kb and encompasses the genes TAP2, DOB, DQB, DQA, DRB, DRA, BTNL2 and a further BTNL gene. The DQ and DR genes of this haplotype are not duplicated. Mimu-DOB is not transcribed and represents a pseudogene due to deletions and premature stop codons. Analysis of BAC clone DNA, a cDNA sample and eight genomic DNA samples suggests that Mimu-DRB, Mimu-DQA and Mimu-DQB are highly polymorphic with the majority of peptide-binding residues being affected by polymorphisms. In contrast, Mimu-DRA is moderately polymorphic, and the variable amino acid positions are not part of the peptide-binding region. Phylogenetic analysis of Mimu-DQA and Mimu-DQB and other primate DQA and DQB genes indicates that duplication of DQA and DQB loci occurred in Anthropoidea after the split from Strepsirrhini.     

Are Dieting and Dietary Inadequacy a Second Hit in the Association with Polycystic Ovary Syndrome Severity?

Background

The composition of the diet is of increasing importance for the development and maturation of the ovarian follicles. In Polycystic Ovary Syndrome (PCOS) healthy dietary interventions improve the clinical spectrum. We hypothesized that dieting and diet inadequacy in the reproductive life course is associated with impaired programming of ovarian follicles and contributes to the severity of the PCOS phenotype.

Methods and Findings

To determine associations between the use of a self-initiated diet and diet inadequacy and the severity of the PCOS phenotype, we performed an explorative nested case control study embedded in a periconception cohort of 1,251 patients visiting the preconception outpatient clinic. 218 patients with PCOS and 799 subfertile controls were selected from the cohort and self-administered questionnaires, anthropometric measurements and blood samples were obtained. The Preconception Dietary Risk Score (PDR score), based on the Dutch dietary guidelines, was used to determine diet inadequacy in all women. The PDR score was negatively associated to cobalamin, serum and red blood cell folate and positively to tHcy. PCOS patients (19.9%), in particular the hyperandrogenic (HA) phenotype (22.5%) reported more often the use of a self-initiated diet than controls (13.1%; p = 0.023). The use of an inadequate diet was also significantly higher in PCOS than in controls (PDR score 3.7 vs 3.5; p = 0.017) and every point increase was associated with a more than 1.3 fold higher risk of the HA phenotype (adjusted OR 1.351, 95% CI 1.09–1.68). Diet inadequacy was independently associated with the anti-Müllerian Hormone (AMH) concentration (β 0.084; p = 0.044; 95% CI 0.002 to 0.165) and free androgen index (β 0.128; p = 0.013; 95% CI 0.028 to 0.229) in PCOS patients.

Conclusions

The use of a self-initiated diet and diet inadequacy is associated with PCOS, in particular with the severe HA phenotype. This novel finding substantiated by the association between diet inadequacy and AMH needs further investigation.     

Effects of prolonged drought on the anatomy of sun and shade needles in young Norway spruce trees

Predicted increases in the frequency and duration of drought are expected to negatively affect tree vitality, but we know little about how water shortage will influence needle anatomy and thereby the trees’ photosynthetic and hydraulic capacity. In this study, we evaluated anatomical changes in sun and shade needles of 20‐year‐old Norway spruce trees exposed to artificial drought stress. Canopy position was found to be important for needle structure, as sun needles had significantly higher values than shade needles for all anatomical traits (i.e., cross‐sectional needle area, number of tracheids in needle, needle hydraulic conductivity, and tracheid lumen area), except proportion of xylem area per cross‐sectional needle area. In sun needles, drought reduced all trait values by 10–40%, whereas in shade needles, only tracheid maximum diameter was reduced by drought. Due to the relatively weaker response of shade needles than sun needles in drought‐stressed trees, the difference between the two needle types was reduced by 25% in the drought‐stressed trees compared to the control trees. The observed changes in needle anatomy provide new understanding of how Norway spruce adapts to drought stress and may improve predictions of how forests will respond to global climate change.     

Plasmids of the pRM/pRF Family Occur in Diverse Rickettsia Species

The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

Different Responses of Norway Spruce Needles from Shaded and Exposed Crown Layers to the Prolonged Exposure to Elevated CO2 Studied by Various Chlorophyll a Fluorescence Techniques

The acclimation depression of capacity of photon utilisation in photochemical reactions of photosystem 2 (PS2) can develop already after three months of cultivation of the Norway spruces (Picea abies [L.] Karst.) under elevated concentrations of CO₂ (i.e., ambient, AC, + 350 µmol(CO₂) mol^–1 = EC) in glass domes with adjustable windows. To examine the role that duration of EC plays in acclimation response, we determined pigment contents, rate of photosynthesis, and parameters of chlorophyll a fluorescence for sun and shade needles after three seasons of EC exposure. We found responses of shaded and exposed needles to EC. Whereas the shaded needles still profited from the EC and revealed stimulated electron transport, for the exposed needles the stimulation of both electron transport activity and irradiance saturated rate of CO₂ assimilation (P _Nmax) under EC already disappeared. No signs of the PS2 impairment were observed as judged from high values of potential quantum yield of PS2 photochemistry (F_V/F_M) and uniform kinetics of Q_A reoxidation for all variants. Therefore, the long-term acclimation of the sun-exposed needles to EC is not necessarily accompanied with the damage to the PS2 reaction centres. The eco-physiological significance of the reported differentiation between the responses of shaded and sun exposed needles to prolonged EC may be in changed contribution of the upper and lower crown layers to the production activity of the tree. Whereas for the AC spruces, P _Nmax of shaded needles was only less than 25 % compared to exposed ones, for the EC spruces the P _Nmax of shaded needles reached nearly 40 % of that estimated for the exposed ones. Thus, the lower shaded part of the crown may become an effective consumer of CO₂.