Neue Fundorte von Orobanchaceen aus dem Mediterrangebiet

Zusammenfassung Es werden bisher noch nicht publizierte Fundorte von Orobanchaceen aus Bulgarien, Südfrankreich, Griechenland, Italien, Jugoslawien, Marokko, Spanien und aus der Türkei angegeben. Eine neue Art,Orobanche Rechingeri, wird beschrieben.Herrn Professor Dr. K. H.Rechinger zum 60. Geburtstag gewidmet.     

Involvement of nitro-compounds in the mutagenicity of urban Pm2.5 and Pm10 in Turin

Fine particles can be active carriers of toxic compounds into the alveoli of the lungs. Among these compounds are numerous mutagens and carcinogens. The direct mutagenicity per unit mass of fine particulate matter (PM) is significantly higher than that of coarse particles, especially in urban areas. In this study, the mutagenic properties of urban PM2.5 and PM10 were evaluated, and the role of nitro-compounds was estimated. PM2.5 and PM10 samplings, and measurements of NOx and some PAHs were performed daily in 2007 in Turin, following a consolidated in vitro test - the Salmonella mutagenicity assay - conducted with organic extracts of PM2.5 and PM10. The mutagenic properties were assessed for each month of sampling with Salmonella typhimurium strain TA98 and TA98-derived strains: a nitroreductase-deficient mutant strain (TA98NR) and an additional nitroreductase-producing plasmid strain (YG1021). The annual measured mean levels of PM2.5 and PM10 were 34±20 and 48±18μg/m(3). The PM2.5/PM10 ratio ranged from 0.36 to 0.89. The Salmonella assay showed higher mutagenicity in autumn/winter (20±15 TA98NR; 54±39 TA98; 173±161 YG1021 net revertants/m(3)) compared with spring/summer (2±2 TA98NR; 7±8 TA98; 24±27 YG1021 net revertants/m(3)) (p<0.01). There are also statistically significant seasonal differences in the gravimetric analysis data. The number of TA98 net revertants per μg of PM2.5 is 6.5 times greater than per μg PM10. Moreover, the bioassay results showed an amplified response in the YG1021 strain and a reduced response in the TA98NR strain. The net revertant ratio TA98NR/YG1021 is 11±4 for organic extracts of PM2.5 and 13±6 for extracts of PM10 (p<0.01). There is a significant correlation between the NOx and PAH concentrations. These findings illustrate the relevant role of nitro compounds, and they underline the priority in improving preventive measures to reduce air pollution by nitrated molecules.     

Thermal Denaturation of Bacterial and Bovine Dihydrofolate Reductases and their Complexes with NADPH,Trimethoprim and Methotrexate

Abstract

Scanning microcalorimetry was used for the study of thermal denaturation of E.coli and bovine liver dihydrofolate reductases (cDHFR and bDHFR, respectively) and their complexes with NADPH, trimethoprim (TMP) and methotrexate (MTX) at pH 6.8. It was shown that the denaturation temperature of bDHFR is 7.2°C less than that of cDHFR and that ionic strength is equally important for the thermostability and cooperativity of the denaturation process of the two proteins. Binding of antifolate compounds significantly stabilizes DHFR against heat denaturation. The stabilizing effect and the transition cooperativity depend on the nature of the inhibitor, the presence of NADPH and the origin of the enzyme. The dependence of calorimetric denaturation enthalpy (calculated per gram of protein) on denaturation temperature for DHFRs, their complexes with NADPH and binary/ternary complexes with TMP/MTX fits to the same straight line with the slope of 0.66 J/K g. This relatively high value indicates an essential role of hydrophobic contacts in the stabilization of DHFR structure. The change of denaturation temperatures in binary complexes with MTX/TMP (in comparison with the free enzymes) is as much as 14.2°C/8.5°C and 13.3°C/3.2°C for cDHFR and bDHFR, respectively. The same change in ternary complexes with MTX/TMP is much more pronounced and equals to 21.9°C/16.8°C and 29.0°C/16.4°C. The vast difference of binary and ternary complexes thermostability demonstrates the important role of cofactor in the stabilization of enzyme. Moving from binary to ternary systems causes a significant increase in denaturation temperatures, even when corresponding association constants do not change (cDHFR binary/ternary complexes with MTX) or increases very slightly (bDHFR binary/ternary complexes with TMP). In all other cases the increase of denaturation temperature     

Intracellular trafficking of FXYD1 (phospholemman) and FXYD7 proteins in Xenopus oocytes and mammalian cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.     

Mutations in LPIN1 cause recurrent acute myoglobinuria in childhood

Recurrent episodes of life-threatening myoglobinuria in childhood are caused by inborn errors of glycogenolysis, mitochondrial fatty acid beta-oxidation, and oxidative phosphorylation. Nonetheless, approximately half of the patients do not suffer from a defect in any of these pathways. Using homozygosity mapping, we identified six deleterious mutations in the LPIN1 gene in patients who presented at 2-7 years of age with recurrent, massive rhabdomyolysis. The LPIN1 gene encodes the muscle-specific phosphatidic acid phosphatase, a key enzyme in triglyceride and membrane phospholipid biosynthesis. Of six individuals who developed statin-induced myopathy, one was a carrier for Glu769Gly, a pathogenic mutation in the LPIN1 gene. Analysis of phospholipid content disclosed accumulation of phosphatidic acid and lysophospholipids in muscle tissue of the more severe genotype. Mutations in the LPIN1 gene cause recurrent rhabdomyolysis in childhood, and a carrier state may predispose for statin-induced myopathy.     

c-Jun N-terminal kinase-3 (JNK3)/stress-activated protein kinase-beta (SAPKbeta) binds and phosphorylates the neuronal microtubule regulator SCG10.

The neuronal growth-associated protein SCG10 is enriched in the growth cones of neurons where it destabilizes microtubules and thus contributes to the dynamic assembly and disassembly of microtubules. Since its microtubule-destabilizing activity is regulated by phosphorylation, SCG10 may link extracellular signals to rearrangements of the neuronal cytoskeleton. To identify signal transduction pathways that may lead to SCG10 phosphorylation, we tested a series of serine-threonine-directed protein kinases that phosphorylate SCG10 in vitro. We demonstrate that purified SCG10 can be phosphorylated by two subclasses of mitogen-activated protein (MAP) kinases, c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) and p38 MAP kinase. Moreover, SCG10 was found to bind tightly and specifically to JNK3/SAPKbeta. JNK3/SAPKbeta phosphorylation occurs at Ser-62 and Ser-73, residues that result in reduced microtubule-destabilizing activity for SCG10. Endogenous SCG10 also undergoes increased phosphorylation in sympathetic neurons at times of JNK3/SAPKbeta activation following deprivation from nerve growth factor. Together these observations indicate that activation of JNK/SAPKs provides a pathway for phosphorylation of SCG10 and control of growth cone microtubule formation following neuronal exposure to cellular stresses.     

Clinical and endocrine characteristics in atypical and classical growth hormone insensitivity syndrome

OBJECTIVE: Classical growth hormone insensitivity syndrome (GHIS) comprises a dysmorphic phenotype, extreme short stature (height SDS < 3), normal GH and low IGF-I and IGFBP-3. Wide clinical variation is recognised with classical and atypical forms. We aimed to delineate features of the milder "atypical" GHIS phenotype, and to determine whether this correlates with milder auxological and biochemical features. METHODS: Fifty-nine patients from a European series of 82 patients with GHIS, with strict diagnostic criteria of GHIS, were studied and assigned to classical or atypical GHIS groups according to facial phenotype, i.e. "classical" required 2 of 3 recognized GHIS features (frontal bossing, mid-facial hypoplasia and depressed nasal bridge), "atypical" required 0 or 1 of these facial features. Classical and atypical GHIS groups were compared in terms of (1) phenotypic features, including high-pitched voice, sparse hair, blue sclera, hypoglycaemia, microphallus, (2) birth length, height SDS, and (3) basal IGF-I, IGF-II, IGFBP-1, IGFBP-3, GHBP and increase in IGF-I on IGF-I generation testing. RESULTS: Fifty patients [24 males, 26 females, aged 8.6 +/- 4.6 years (mean +/- SD)] had "classical GHIS", 9 patients (7 males, 2 females, aged 7.8 +/- 4.1 years) had "atypical GHIS", 7 with normal facies. Atypical GHIS patients had lesser height deficit (Ht SDS -4.0 +/- 1.4) compared to classical GHIS (-6.7 +/- 1.4), less reduction in IGFBP-3 SDS (atypical -5.5 +/- 3.3; classical -8.6 +/- 2.4), and more had normal GHBP (>10% binding). Other variables were also less frequent in atypical GHIS patients: high-pitched voice 11% (70% classical), sparse hair 11% (42% classical), blue sclera 0% (38% classical), hypoglycaemia 11% (42% classical), and microphallus 14% (1 of 7 males), compared to 79% of classical (19 of 24 males). CONCLUSIONS: Atypical GHIS patients, with relatively normal facial appearance, demonstrate less height defect and biochemical abnormalities compared to classical patients. GH insensitivity may be present in children with short stature and an otherwise normal appearance.     

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.