Competition of Pyridoxal 5'-Phosphate with Ribulose 1,5-Bisphosphate and Effector Sugar Phosphates at the Reaction Centers of the Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The Stimulation of the carboxylase reaction by effectors of ribulose 1,5-bisphosphate carboxyl­ ase/oxygenase displays higher sensitivity towards pyridoxal 5'-phosphate inhibition than the catalytical process itself. Pyridoxal 5'-phosphate binding to the enzyme is not affected by the modulators 6-phospho-gluconate and fructose 1,6-bisphosphate at low concentrations at which these agents stimulate the carboxylation rate. At higher concentrations these sugar phosphates protect the enzyme against pyridoxal 5'-phos-phate inhibition in a similar fashion like the substrate ribulose 1,5-bisphosphate. Such protection experiments in combination with spectrophotometrical studies of pyridoxal 5'-phosphate binding demonstrate two binding states of ribulose 1,5-bisphosphate at the reaction centers of the enzyme with different requirements for Mg2+. 6-Phosphogluconate functions as protector only in the presence of Mg2+. Our results imply a competition between pyridoxal 5'-phosphate and substrate or effector sugar phosphates at the reaction centers of the spinach carboxylase. It is proposed that the pyridoxal 5'-phosphate inhibition of the stimulatory activity of these effectors originates from a modification of the regulatory sites of the enzyme caused by pyridoxal 5'-phosphate binding to the catalytical sites

Characteristic Features of the Regulatory Functions of the ᴅ-Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Spinach

Catalysis and regulation of CO2 fixation differ in a characteristic manner in their response to anionic modifiers and the polarity of the reaction medium. Monovalent inorganic anions inhibit catalysis and CO2-activation of the ᴅ-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach, whereas the activity and binding of NADPH and effector sugar phosphates are affected only at appreciably higher concentrations. In contrast such modulators with a dianion structure stimulate CO2 fixation by an increase of the affinity of the enzyme for the activator CO2 and stabilization of the reactive carbamate. Structure-activity studies revealed a broad specificity of the enzyme for these regulatory effects. Essentially amino groups are involved in these processes. Certain organic solvents, as methanol or acetone, stimulate CO2 fixation by a similar modification of the CO2 activation centers, as induced by dianionic effectors. These results infer that such effects are due to a decrease of the polarity at the regulatory centers of the enzyme and a concomitant change of the pK of the active lysine responsible for the binding of the activator CO2. A correlation of effector binding and activity demonstrates that already low, non-saturating concentrations of such modifiers induce high activation levels of the carboxylase and prevent the dissociation of the activated ternary complex. It is discussed that the central problem concerning the catalytical competence of ᴅ-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the presence of active site directed dianionic effectors can be solved kinetically

Regulation of stanniocalcin-1 secretion by BeWo cells and first trimester human placental tissue from normal pregnancies and those at increased risk of developing preeclampsia.

Stanniocalcin-1 (STC-1) is a multi-functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC-1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto-maternal interface is unknown. Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC-1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC-1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC-1 but it required co-stimulation with the Adenosine-3', 5'-cyclic monophosphate (cAMP) analogue, 8-Bromo adenosine-3', 5'-cyclic monophosphate cAMP (8 Br-cAMP) to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF-2α) and the Phosphatidylinositol-3 kinase (PI3 -Kinase)/AKT/Serum and glucocorticoid-induced kinase-1(SGK-1) pathway resulted in significant inhibition of STC-1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC-1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia

Enhanced overexpression of an HIF-1/hypoxia-related protein in cancer cells.

Cap43 is a protein whose RNA is induced under conditions of severe hypoxia or prolonged elevations of intracellular calcium. Additionally, Ni and Co also induce Cap43 because they produce a state of hypoxia in cells. Cap43 protein is expressed at low levels in normal tissues; however, in a variety of cancers, including lung, brain, melanoma, liver, prostate, breast, and renal cancers, Cap43 protein is overexpressed in cancer cells. The low level of expression of Cap43 in some normal tissues compared with their cancerous counterparts, combined with the high stability of Cap43 protein and mRNA, makes the Cap43 gene a new, important cancer marker. We hypothesize that the mechanism of Cap43 overexpression in cancer cells involves a state of hypoxia characteristic of cancer cells where the Cap43 protein becomes a signature for this hypoxic state

Interazione del Ni(II) e del Cu(II) con la sequenza N-terminale dell’istone H4

I composti del nichel sono ben conosciuti come agenti cancerogeni per l'uomo. I meccanismi molecolari responsabili della tossicità e cancerogenesi del nichel coinvolgono: danni promutagenici al DNA, ossia danni ossidativi alle nucleo-basi; effetti epigenetici sulla cromatina, il nichel interferisce con la struttura della cromatina, compattazione di essa, metilazione, acetilazione e quindi con la trascrizione. Recenti studi hanno rivelato che non tutti i composti del nichel sono ugualmente tossici: i sali acquo-insolubili mostrano una potente attività, mentre quelli solubili ne mostrano una minore. La ragione dell'alta attività dei composti acquo-insolubili è dovuta alla loro capacità di penetrare nelle cellule attraverso un processo di fagocitosi che rappresenta una maniera molto efficiente per l'accumulo dello ione metallico all'interno delle cellule. Cellule epiteliali lungo l'apparato respiratorio sono in grado di fagocitare composti insolubili del nichel e discioglierli nell'ambiente acido dei vacuoli, fornendo una continua sorgente di ioni Ni(II) con conseguenti alti livelli intracellulari

Exhaled Breath Condensate as a Suitable Matrix to Assess Lung Dose and Effects in Workers Exposed to Cobalt and Tungsten

The aim of the present study was to investigate whether exhaled breath condensate (EBC), a fluid formed by cooling exhaled air, can be used as a suitable matrix to assess target tissue dose and effects of inhaled cobalt and tungsten, using EBC malondialdehyde (MDA) as a biomarker of pulmonary oxidative stress. Thirty-three workers exposed to Co and W in workshops producing either diamond tools or hard-metal mechanical parts participated in this study. Two EBC and urinary samples were collected: one before and one at the end of the work shift. Controls were selected among nonexposed workers. Co, W, and MDA in EBC were analyzed with analytical methods based on mass spectrometric reference techniques. In the EBC from controls, Co was detectable at ultratrace levels, whereas W was undetectable. In exposed workers, EBC Co ranged from a few to several hundred nanomoles per liter. Corresponding W levels ranged from undetectable to several tens of nanomoles per liter. A parallel trend was observed for much higher urinary levels. Both Co and W in biological media were higher at the end of the work shift in comparison with preexposure values. In EBC, MDA levels were increased depending on Co concentration and were enhanced by coexposure to W. Such a correlation between EBC MDA and both Co and W levels was not observed with urinary concentration of either element. These results suggest the potential usefulness of EBC to complete and integrate biomonitoring and health surveillance procedures among workers exposed to mixtures of transition elements and hard metals

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1