Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization

Friedreich ataxia (FA) Is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (beta-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and beta-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis. (C) 2002 by The American Society of Hematology.

The role of the Eph-ephrin signalling system in the regulation of developmental patterning

The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.

Body mass index-related human adipocyte agouti expression is sex-specific but not depot-specific

Objective: To determine if human adipocyte agouti signal protein (ASIP) mRNA expression is associated with obesity and is gender and/or depot specific. Research Methods and Procedures: Subjects included 8 men (64 +/- 3 years) and 14 women (56 +/- 15 years) undergoing elective abdominal surgery. ASIP mRNA levels in isolated omental and subcutaneous abdominal adipocytes were measured by quantitative reverse transcription polymerase chain reaction. Results: No significant depot difference was observed between genders; ASIP mRNA levels of omental and subcutaneous abdominal adipocytes were pooled for this analysis. BMI and ASIP gene expression were negatively correlated in men (p = -0.70; p < 0.05), whereas a positive relationship was observed in women (p = 0.48; p < 0.05). No significant difference was observed in age, body weight, body mass index (BMI), and waist circumference between groups. Hip circumference was significantly higher in women than in men (p < 0.05). Also, no significant difference in ASIP mRNA expression was observed between men and women, regardless of the fat depot. Discussion: These results show that men and women of similar age and BMI present similar ASIP mRNA levels in omental and subcutaneous abdominal adipocytes. However, a sexual dimorphism exists in the relationship between ASIP expression and BMI. If ASIP is involved in appetite regulation or energy homeostasis in humans, this observation may contribute to the recognized differences in these parameters between men and women.

PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity

PrrC from Rhodobacter sphaeroides provides the signal input to a two-component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry, 39, 2052-2062; Oh, J.-I. and Kaplan, S. (2000) EMBO J. 19, 42374247). It is also a homologue of eukaryotic Sco proteins and each has a C-x-x-x-C-P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis Of the Cu-A centre of respiratory cytochrome oxidase. Overexpression and purification of a water-soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal-free rPrrC (apo-PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV-visible spectrum, having absorbance with a lambda(max) of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal-free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O-2 sensitivity are involved in copper binding in reduced PrrC since the same copper-loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol-disulfide oxidoreductase function and a copper-binding role. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasl::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604,2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket or Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2 mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2 mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24 h, The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity, Conversely, pro-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action. (C) 2002 Elsevier Science Ltd. All rights reserved.

Future contributions of crop modelling - from heuristics and supporting decision making to understanding genetic regulation and aiding crop improvement

Crop modelling has evolved over the last 30 or so years in concert with advances in crop physiology, crop ecology and computing technology. Having reached a respectable degree of acceptance, it is appropriate to review briefly the course of developments in crop modelling and to project what might be major contributions of crop modelling in the future. Two major opportunities are envisioned for increased modelling activity in the future. One opportunity is in a continuing central, heuristic role to support scientific investigation, to facilitate decision making by crop managers, and to aid in education. Heuristic activities will also extend to the broader system-level issues of environmental and ecological aspects of crop production. The second opportunity is projected as a prime contributor in understanding and advancing the genetic regulation of plant performance and plant improvement. Physiological dissection and modelling of traits provides an avenue by which crop modelling could contribute to enhancing integration of molecular genetic technologies in crop improvement. Crown Copyright (C) 2002 Published by Elsevier Science B.V. All rights reserved.

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PCH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20% with glucose loading. Cot-relation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not Suppressed by oral glucose loading in non-diabetic mothers.

A prostaglandin F2a Analog induces suppressors of the cytokine signalling-3 expression n the corpus luteum of the pregnant rat: A potential new mechanism in luteolysis

PRL and placental lactogen (PL) play key roles in maintaining the rodent corpus luteum through pregnancy. Suppressors of cytokine signaling (SOCS) have been shown to decrease cell sensitivity to cytokines, including PRL, and so here we have addressed the issue of whether luteolysis induced by prostaglandin F-2alpha (PGF(2alpha)) might up-regulate SOCS proteins to inhibit PRL signaling. In d 19 pregnant rats, cloprostenol, a PGF(2alpha) analog, rapidly induced transcripts for SOCS-3 and, to a lesser extent, SOCS-1. We also found increased SOCS-3 protein in the ovary by immunoblot and in the corpus luteum by immunohistochemistry. Increased SOCS-3 expression was preceded by an increase in STAT3 tyrosine phosphorylation 10 min after cloprostenol injection and was maintained for 4 h, as determined by gel shift and immunohistochemistry. Induction of SOCS-3 was accompanied by a sharp decrease in active STAT5, as determined by gel-shift assay and by loss of nuclear localized STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to down-regulation of PRL receptor. Therefore, induction of SOCS-3 by PGF(2alpha) may be an important element in the initiation of luteolysis via rapid suppression of luteotropic support from PL.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone

The renal sodium-sulfate cotransporter, NaSi-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) and tri-iodothyronine (T-3). Recently, we cloned the mouse NaSi-1 gene (Nas1) and in the present study identified a 1,25-(OH)(2)D-3- and T-3-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3-responsive element (IR0 T3RE) at -436 to -425 which conferred 1,25(OH)(2)D-3 and T3 responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D-3 and T-3, and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Indirect evidence of density-dependent population regulation in Aponomma hydrosauri (Acari : Ixodidae), an ectoparasite of reptiles

The extent to which density-dependent processes regulate natural populations is the subject of an ongoing debate. We contribute evidence to this debate showing that density-dependent processes influence the population dynamics of the ectoparasite Aponomma hydrosauri (Acari: Ixodidae), a tick species that infests reptiles in Australia. The first piece of evidence comes from an unusually long-term dataset on the distribution of ticks among individual hosts. If density-dependent processes are influencing either host mortality or vital rates of the parasite population, and those distributions can be approximated with negative binomial distributions, then general host-parasite models predict that the aggregation coefficient of the parasite distribution will increase with the average intensity of infections. We fit negative binomial distributions to the frequency distributions of ticks on hosts, and find that the estimated aggregation coefficient k increases with increasing average tick density. This pattern indirectly implies that one or more vital rates of the tick population must be changing with increasing tick density, because mortality rates of the tick's main host, the sleepy lizard, Tiliqua rugosa, are unaffected by changes in tick burdens. Our second piece of evidence is a re-analysis of experimental data on the attachment success of individual ticks to lizard hosts using generalized linear modelling. The probability of successful engorgement decreases with increasing numbers of ticks attached to a host. This is direct evidence of a density-dependent process that could lead to an increase in the aggregation coefficient of tick distributions described earlier. The population-scale increase in the aggregation coefficient is indirect evidence of a density-dependent process or processes sufficiently strong to produce a population-wide pattern, and thus also likely to influence population regulation. The direct observation of a density-dependent process is evidence of at least part of the responsible mechanism.