Molecular characterization of an Arabidopsis gene encoding a phospholipid-specific inositol polyphosphate 5-phosphatase

Phosphoinositides are important molecules that serve as second messengers and bind to a complex array of proteins modulating their subcellular location and activity. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. We previously reported the identification of 15 putative 5PTase genes in Arabidopsis and have shown that overexpression of the At5PTase1 gene can alter abscisic acid signaling. At5PTase1 and At5PTase2 have been shown to hydrolyze the 5-phosphate from inositol phosphate substrates. We have examined the substrate specificity of the At5PTase11 protein, which is one of the smallest predicted 5PTases found in any organism. We report here that the At5PTase11 gene encodes an active 5PTase enzyme that can only dephosphorylate phosphoinositide substrates containing a 5-phosphate. In addition to hydrolyzing known substrates of 5PTase enzymes, At5PTase11 also hydrolyzes the 5-phosphate from phosphatidylinositol (3,5) bisphosphate. We also show that the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways.     

Long-term follow-up of total penile reconstruction with sensate osteocutaneous free fibula flap in 18 biological male patients

Surgical reconstruction of the penis is challenging because of the many cosmetic and functional (e.g., sexual intercourse and voiding) requirements that must be addressed. Since the free sensate osteocutaneous fibula flap was first described for total penile reconstruction in 1993 it has been widely accepted, with its advantages and minimal shortcomings. In this article, the authors present the longest follow-up of biologically male patients with free fibular phalloplasties. Since 1994, 18 biologically male patients with total penile losses for various reasons were treated with free sensate osteocutaneous fibula flaps. All patients were included in the study. The ages of the patients ranged between 20 and 26 years (mean, 22.2 years). The average follow-up period was 5.4 years (range, 1 to 9 years). Patient satisfaction was evaluated by a questionnaire regarding both quality of orgasm and daily activities. Conventional radiographic imaging, magnetic resonance imaging, and bone mineral densitometry were performed to evaluate the fate of the bony component of the flap. Also, sensibility was evaluated by bulbocavernous reflex and penile somatosensory evoked potentials testing in nine patients. Six patients married, and five of them had six children. Most patients and their partners reported pleasurable sexual intercourse and orgasm. Conventional radiographs of the fibular bone in neophalluses showed robust, calcified bone structure without any evidence of bone resorption or fracture. The magnetic resonance images showed the cortical substance and spongiosum of the bone marrow, which are characteristic signs of bone viability. After intravenous injection of gadolinium, the neophallus bone showed uptake of contrast medium. Viability of neophallus bone was shown even at 9-year follow-up (the longest follow-up in the literature). Dual energy x-ray absorptiometry measurements of the penile bone grafts showed that fibular components in the penis had bone mineral density values that were close to but lower than those of intact fibula in the same subjects. These results were considered as evidence of viability of bone grafts. Neural integrity was found between the nerves of the neophallus and the residual penile bodies by both bulbocavernous reflex and penile somatosensory evoked potentials tests. In conclusion, free sensate fibula flap phalloplasty provides the cosmetic and functional requirements that an ideal penis should have. All results put an end to the discussion that the fibular component of the neophallus could resorb. Constitution of neural integrity is important in terms of pleasurable sexual intercourse. The authors believe the free sensate osteocutaneous fibula flap should be considered as the standard in penile reconstruction.     

D-Serine as a putative glial neurotransmitter

Abundant recent evidence favors a neurotransmitter/neuromodulator role for D-serine. D-serine is synthesized from L-serine by serine racemase in astrocytic glia that ensheath synapses, especially in regions of the brain that are enriched in NMDA-glutamate receptors. D-serine is more potent than glycine at activating the 'glycine' site of these receptors. Moreover, selective degradation of D-serine but not glycine by D-amino acid oxidase markedly reduces NMDA neurotransmission. D-serine appears to be released physiologically in response to activation by glutamate of AMPA-glutamate receptors on D-serine-containing glia. This causes glutamate-receptor-interacting protein, which binds serine racemase, to stimulate enzyme activity and D-serine release. Thus, glutamate triggers the release of D-serine so that the two amino acids can act together on postsynaptic NMDA receptors. D-serine also plays a role in neural development, being released from Bergmann glia to chemokinetically enhance the migration of granule cell cerebellar neurons from the external to the internal granular layer.     

Thermodynamic phase plane analysis of ventricular contraction and relaxation

Background  

Ventricular function has conventionally been characterized using indexes of systolic (contractile) or diastolic (relaxation/stiffness) function. Systolic indexes include maximum elastance or equivalently the end-systolic pressure volume relation and left ventricular ejection fraction. Diastolic indexes include the time constant of isovolumic relaxation - and the end-diastolic pressure-volume relation. Conceptualization of ventricular contraction/relaxation coupling presents a challenge when mechanical events of the cardiac cycle are depicted in conventional pressure, P, or volume, V, terms. Additional conceptual difficulty arises when ventricular/vascular coupling is considered using P, V variables.     

Postnatal glucocorticoids induce alpha-ENaC formation and regulate glucocorticoid receptors in the preterm rabbit lung

At birth, lung fluid clearance is coupled to Na+ transport through epithelial Na+ channels (ENaC) in the distal lung epithelium. We evaluated the effect of postnatal glucocorticoids (GC) on lung alpha-ENaC expression in preterm 29-day gestational age (GA) fetal rabbits. Postnatal treatment of 29-day GA fetuses with 0.5 mg/kg of dexamethasone (Dex) iv resulted in a 2- and 22-fold increase in lung alpha-ENaC mRNA expression compared with saline-treated fetuses after 8 and 16 h, respectively. Lung alpha-ENaC protein levels in Dex-treated fetuses were also elevated compared with saline-treated counterparts. The extravascular lung water (EVLW)/dry lung tissue weight ratios of 29-day GA fetuses treated with either saline or Dex decreased over 24 h compared with that observed at birth; however, at 24 h, the EVLW/dry lung tissue weight ratios of saline- and Dex-treated fetuses were similar. Dex-induced alpha-ENaC mRNA and protein levels were attenuated by glucocorticoid receptor (GCR) antagonist RU-486 in fetal distal lung epithelial cells isolated from 29-day GA fetuses, indicating that GC-dependent augmentation of lung alpha-ENaC requires the presence of functional GCR. Lung GCR mRNA expression and protein levels were elevated in 29-day GA fetuses compared with fetuses at earlier GA. Exposure of 29-day GA fetuses to Dex for 16 h caused a 2.1-fold increase in lung GCR mRNA expression, but GCR protein levels were decreased in Dex-treated fetuses after 24 h. We conclude that postnatal treatment of preterm 29-day GA fetal rabbits with GC results in an elevation of lung alpha-ENaC accompanied by an autoregulation of pulmonary GCR.     

Arabidopsis thaliana Ogg1 protein excises 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from oxidatively damaged DNA containing multiple lesions

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.     

Cytotoxic factor-autoantibodies: possible role in the pathogenesis of dengue haemorrhagic fever

During dengue virus infection a unique cytokine, cytotoxic factor (hCF), is produced that is pathogenesis-related and plays a key role in the development of dengue haemorrhagic fever (DHF). However, what regulates the adverse effects of hCF is not known. We have previously shown that anti-hCF antibodies raised in mice, neutralise the pathogenic effects of hCF. In this study we have investigated the presence and levels of hCF-autoantibodies in sera of patients with various severity of dengue illness (n=136) and normal healthy controls (n=50). The highest levels of hCF-autoantibodies (mean+/-S.D.=36+/-20 U ml(-1)) were seen in patients with mild illness, the dengue fever (DF), and 48 out of 50 (96%) of the sera were positive. On the other hand the hCF-autoantibody levels declined sharply with the development of DHF and the levels were lowest in patients with DHF grade IV (mean+/-S.D.=5+/-2 U ml(-1); P=<0.001 as compared to DF). Only one of the 13 DHF grade IV patients had an antibody level above the 'cut-off' value (mean plus 3 S.D. of the control sera). The analysis of data with respect to different days of illness further showed that the highest levels of hCF-autoantibodies were present in DF patients at >9 days of illness. Moreover, the DF patients at all time points, i.e. 1-4, 5-8 and >9 days of illness had significantly higher levels of hCF-autoantibodies (P<0.001) than patients with DHF grade I, II, III and IV. In addition DHF grade I and grade II patients had significantly more positive specimens than DHF grade III and grade IV patients at all time points. These results suggest that elevated levels of hCF-autoantibodies protect the patients against the development of severe forms of DHF and, therefore, it may be useful as a prognostic indicator.     

Flavonoid glycosides from Centaurea pseudoscabiosa subsp. pseudoscabiosa from Turkey

Three flavonoid glycosides were isolated and characterized, together with a further 13 substances belonging to various classes of compounds, in particular two phenolic acids, a coumarin, a sugar and nine flavonoids from the flowered aerial parts of Centaurea pseudoscabiosa subsp. pseudoscabiosa Boiss. et Buhse (Asteraceae). Some considerations about their evolutionary meaning are provided.     

Poly(alpha-alkyl gamma-glutamate)s of microbial origin. 2. On the microstructure and crystal structure of poly(alpha-ethyl gamma-glutamate)s

The stereochemical microstructure and crystalline structure of nearly racemic poly(alpha-ethyl gamma,DL-glutamate) obtained by esterification of biosynthetic poly(gamma-glutamic acid) were examined by NMR, DSC, and powder X-ray diffraction. The two enantiomerically pure poly(alpha-ethyl gamma-glutamate)s, as well as the racemic stereocopolymers with random and alternating microstructure, were prepared by chemical synthesis and studied in parallel to help in the interpretation of the data. The (13)C NMR analysis revealed that biosynthetic poly(alpha-ethyl gamma,DL-glutamate) consists of a block stereocopolymer accompanied by minor amounts of a mixture of the two optically pure homopolymers. The polymer is crystalline, with a degree of crystallinity and crystal structure essentially similar to those displayed by the optically pure polymers but clearly different from the alternating copolymer. Conversely, the racemic stereocopolymer with a random microstructure prepared by chemical synthesis is amorphous. The crystal structure of the racemic mixture of the D- and L-homopolymers seems to be very close to that of the biosynthetic stereocopolymer, although some indications suggesting the existence of a stereocomplex were found.     

Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout,coho and Atlantic salmon

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.