Metabolism of α-aminoisobutyric acid in mungbean hypocotyls in relation to metabolism of 1-aminocyclopropane-1-carboxylic acid

1-Aminocyclopropane-1-carboxylic acid (ACC) is known to be converted to ethylene and conjugated into N-malonyl-ACC in plant tissues. When -amino[1-¹⁴C]isobutyric acid (AIB), a structural analog of ACC, was administered to mungbean (Vigna radiata L.) hypocotyl segments, it was metabolized to ¹⁴CO₂ and conjugated to N-malonyl-AIB (MAIB). -Aminoisobutyric acid inhibited the conversion of ACC to ethylene and also inhibited, to a lesser extent, N-malonylation of ACC and d-amino acids. Although the malonylation of AIB was strongly inhibited by ACC as well as by d-amino acids, the metabolism of AIB to CO₂ was inhibited only by ACC but not by d-amino acids. Inhibitors of ACC conversion to ethylene such as anaerobiosis, 2,4-dinitrophenol and Co²⁺, similarly inhibited the conversion of AIB to CO₂. These results indicate that the malonyalation of AIB to MAIB is intimately related to the malonylation of ACC and d-amino acids, whereas oxidative decarboxylation of AIB is related to the oxidative degradation of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyric acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid - MAIB -(malonylamino)-isobutyric acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Patterns of associations of clinical features in neurofibromatosis 1 (NF1)

Neurofibromatosis 1 (NF1) is a common, fully penetrant autosomal dominant disease. The clinical course is generally progressive but highly variable, and the pathogenesis is poorly understood. We studied statistical associations among 13 of the most common or important clinical features in data from four separate sets of NF1 patients: a "developmental sample" of 1,413 probands from the NNFF International Database, an independent "validation sample" of 1,384 probands from the same database, 511 affected relatives of these probands, and 441 patients from a population-based registry in northwest England. We developed logistic regressive models for each of the 13 features using the developmental sample and attempted to validate these models in the other three samples. Age and gender were included as covariates in all models. Models were successfully developed and validated for ten of the 13 features analysed. The results are consistent with grouping nine of the clinical features into three sets: (1) café-au-lait spots, intertriginous freckling and Lisch nodules; (2) cutaneous, subcutaneous and plexiform neurofibromas; (3) macrocephaly, optic glioma and other neoplasms. In addition, three-way interactions among café-au-lait spots, intertriginous freckling and subcutaneous neurofibromas indicate that the first two groups are not independent. Our studies show that some individuals with NF1 are more likely than others to develop certain clinical features of the disease. Some NF1 features appear to share pathogenic mechanisms that are not common to all features.     

Glycosylation ofα 1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of ₁-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewis^x (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.Abbreviations AAL Aleuria aurantia lectin - AGP ₁-acid glycoprotein - CAIE crossed affinoimmunoelectrophoresis - Con A Concanavalin A - HSPC human serum protein calibrator - IL-1 interleukin 1 - IL-6 interleukin 6 - LIF leukaemia inhibitory factor - LPS lipopolysaccharide - SLeX sialyl Lewis^x - TNF tumour necrosis factor     

Pathogenic role of HIF-1α in prostate hyperplasia in the presence of chronic inflammation

Benign prostatic hyperplasia (BPH) commonly occurs in older men with chronic prostatitis. Although BPH is frequently accompanied by inflammation, it is unclear whether inflammation underlies prostate enlargement. Recently, we reported that hypoxia‐inducible factor 1α (HIF-1α), which is known to be induced by proinflammatory cytokines, is involved in testosterone-induced prostate hyperplasia. Therefore, we hypothesized that cytokines secreted from infiltrated macrophages under inflammatory conditions stimulate prostate enlargement by up‐regulating HIF-1α. In the present study, we injected lipopolysaccharide (LPS) into rat prostates to mimic prostatitis and evaluated prostate hyperplasia 14 days later. Epithelial cells of LPS-treated prostates were found to be highly proliferative and HIF-1α levels in prostate tissues to be elevated. When prostate epithelial cells were incubated in conditioned medium from macrophages activated with LPS, they robustly expressed HIF-1α, and under these conditions IL-1β, IL-6, and TNF-α cytokines were found to mediate HIF-1α induction. In addition, HIF-1α was found to enhance the expression of Twist, which initiates epithelial–mesenchymal transition (EMT). Furthermore, profound EMT features were observed in LPS-treated rat prostates, and the natural HIF-1α inhibitors ascorbate and curcumin were found to attenuate EMT and prostate hyperplasia both in vivo and in vitro. Based on these results, we propose that HIF-1α mediates prostate enlargement under inflammatory conditions, and we suggest that HIF-1α be viewed as a promising target for blocking the transition from prostatitis to BPH.     

Regulation of biosynthesis of 1-acetyl-β-carboline in Streptomyces kasugaensis

Summary Biosynthesis of 1-acetyl--carboline, an enzyme inhibitor, in S. kasugaensis SK-619 was regulated by quality of the carbon source and by addition of organic acids into medium. Variation in phosphate concentration also affected production of this carboline.     

Modular Evolution of PGC-1α in Vertebrates

In mammals, the peroxisome proliferator activated receptor (PPAR)γ coactivator-1α (PGC-1α) is a central regulator of mitochondrial gene expression, acting in concert with nuclear respiratory factor-1 (NRF-1) and the PPARs. Its role as a “master regulator” of oxidative capacity is clear in mammals, but its role in other vertebrates is ambiguous. In lower vertebrates, although PGC-1α seems to play a role in coordinating the PPARα axis as in mammals, it does not appear to be involved in NRF-1 regulation of mitochondrial content. To evaluate the evolutionary patterns of this coactivator in fish and mammals, we investigated the evolutionary trajectories of PGC-1α homologs in representative vertebrate lineages. A phylogeny of the PGC-1 paralogs suggested that the family diversified through repeated genome duplication events early in vertebrate evolution. Bayesian and maximum likelihood phylogenetic reconstructions of PGC-1α in representative vertebrate species revealed divergent evolutionary dynamics across the different functional domains of the protein. Specifically, PGC-1α exhibited strong conservation of the activation/PPAR interaction domain across vertebrates, whereas the NRF-1 and MEF2c interaction domains experienced accelerated rates of evolution in actinopterygian (fish lineages) compared to sarcopterygians (tetrapod lineages). Furthermore, analysis of the amino acid sequence of these variable domains revealed successive serine- and glutamine-rich insertions within the teleost lineages, with important ramifications for PGC-1α function in these lineages. Collectively, these results suggest modular evolution of the PGC-1α protein in vertebrates that could allow for lineage-specific divergences in the coactivating capabilities of this regulator.     

Participation of C6C1 unit in the biosynthesis of ephedrine in Ephedra

Feeding of benzoic acid-[7-¹⁴C], benzaldehyde-[7-¹⁴C] and cinnamic acid-[3-¹⁴C] to Ephedra distachya resulted in efficient incorporations of ¹⁴C into the α-carbon atom of the side chain of l-ephedrine. Thus ephedrine was shown to be biosynthesized by the condensation of a C₆C₁ portion which is derived from phenylalanine via cinnamate and an unidentified C₂-N fragment.     

Plasma levels of TGF-β1 in homeostasis of the inflammation in sickle cell disease

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-β), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-β1, and also checked the influence of the presence of −509C/T polymorphism in TGFB1 gene on TGF-β1 plasma values. The plasma levels of TGF-β1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sβ-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The −509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-β1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-β1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-β1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-β1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.     

Selective Abortion of Two Nonsister Nuclei in a Developing Ascus of the hfd1–1 Mutant in SACCHAROMYCES CEREVISIAE

A recessive mutation, hfd1–1, in strain SOS4 of Saccharomyces cerevisiae leads the mutant cells to produce predominantly two-spored asci. Light microscopical examination of Giemsa-stained cells revealed no significant differences in the meiotic figures between mutant and wild-type strains. However, only two of the four meiotic products in a developing ascus matured to ascospores in SOS4. Dyad analysis was carried out on an hfd1–1 mutant strain heterozygous for three markers, asp5, gal1 and arg4, which are closely linked to their centromeres, and for his4, which is loosely linked to its centromere. The twospored asci produced by the hfd1–1 mutant segregated dominant (+) and recessive (-) alleles of each marker in a 1:1 ratio; they generally contained one + and one - spore for any given marker. The occurrence of rare dyads with two + or two - spores can be explained quantitatively by recombination between the marker and its centromere. From the results of these cytological and genetical analyses, we infer that, in the mutant strain, one genome set is partitioned to each of the four second-meiotic division poles, but only two nonsister genomes are incorporated into mature spores. Thus, the hfd1–1 mutation in SOS4 blocks incorporation of two nonsister nuclei into mature ascospores, but does not block enclosure of the remaining two nonsister nuclei.     

Role of PGC-1α in Mitochondrial Quality Control in Neurodegenerative Diseases

Neurochemical Research - As one of the major cell organelles responsible for ATP production, it is important that neurons maintain mitochondria with structural and functional integrity; this is...     

Expression of KiSS-1 in rat oviduct: possible involvement in prevention of ectopic implantation?

The mammalian oviduct is a crucial site for essential postovulatory events in the female reproductive system. These events are, in part, accomplished by clear-cut oviductal segmentation, which helps to provide appropriate epithelial and fluid microenvironments. Early embryonic development and the timely transport of the embryo to the uterus must be promoted, but implantation within the oviduct itself must be avoided. Indeed, the rarity of extra-uterine pregnancies in laboratory animals strongly suggests that active mechanisms operate to prevent ectopic implantation. Kisspeptins, products of the KiSS-1 gene, have been proposed as physiological regulators of uterine implantation by limiting the invasion of the trophoblast into the maternal decidua. We describe here the patterns of expression of the KiSS-1 gene and of kisspeptin immunoreactivity (IR) in the rat oviduct. KiSS-1 mRNA is readily detectable in oviduct samples from all phases of the estrous cycle, whereas kisspeptin-IR is detected in rat oviduct with a regionalized pattern of distribution, viz., strong expression in the isthmus, faint signals in the proximal ampulla, and a lack of immunostaining in the fimbriated infundibulum and interstitial portion. When positive, IR has been localized at the adluminal surface and the cytoplasmic domain of secretory cells. Of note, KiSS-1 expression (at the mRNA and protein levels) shows cycle-related changes with peak expression in proestrus/estrus and lower levels at metestrus/diestrus. This knowledge of the regional- and cycle-specific pattern of expression of KiSS-1 in rat oviduct should open up the possibility of a physiological role of kisspeptins in the prevention of ectopic (tubal) implantation. This work was supported by grants BFU 2005-01443 and BFU 2005-07446 (Ministerio de Educacion y Ciencia, Spain), by funds from the Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and by EU research contract EDEN QLK4-CT-2002-00603. M.T.-S. is also supported by grants from the CIBER Fisiopatología de la Obesidad y Nutrición (CB06/03/0003; Instituto de Salud Carlos III) of which he is a member.     

Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed ⁴C₁ chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme?s catalytic triad (Asp¹⁹⁶, Glu²³⁷ and Asp²⁹⁵) and from Arg¹³⁷. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe⁵², a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe⁵² by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp¹⁹⁶) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.     

Role of transglutaminase 1 in stabilisation of intercellular junctions of the vascular endothelium

Microvascular endothelial monolayers from mouse myocardium (MyEnd) cultured for up to 5 days postconfluency became increasingly resistant to various barrier-compromising stimuli such as low extracellular Ca²⁺ and treatment with the Ca²⁺ ionophore A23187 and with the actin depolymerising compound cytochalasin D. In contrast, microvascular endothelial monolayers from mouse lung microvessels (PulmEnd) remained sensitive to these conditions during the entire culture period which corresponds to the well-known in vivo sensitivity of the lung microvasculature to Ca²⁺ depletion and cytochalasin D treatment. One molecular difference between pulmonary and myocardial endothelial cells was found to be transglutaminase 1 (TGase1) which is strongly expressed in myocardial endothelial cells but is absent from pulmonary endothelial cells. Resistance of MyEnd cells to barrier-breaking conditions correlated strongly with translocation of TGase1 to intercellular junctions. Simultaneous inhibition of intracellular and extracellular TGase activity by monodansylcadaverine (MDC) strongly weakened barrier properties of MyEnd monolayers, whereas inhibition of extracellular TGases by the membrane-impermeable active site-directed TGase inhibitor R281 did not reduce barrier properties. Weakening of barrier properties could be also induced in MyEnd cells by downregulation of TGase1 expression using RNAi-based gene silencing. These findings suggest that crosslinking activity of intracellular TGase1 at intercellular junctions may play a role in controlling barrier properties of endothelial monolayers.