Two proteins were isolated, in a stable form, from bovine brain by ion exchange chromatography, gel filtration and ultracentrifugation on glycerol gradient. They were identified as 20S and 26S ...proteasomes on the basis of molecular mass, migration velocity on non-denaturing gels, immunoreactivity, multipeptidase activity and the 26S proteasome also for dependence on ATP for the degradation of short peptides and ubiquitinylated proteins. However, the 26S proteasome has some properties not yet described for its counterpart of other tissues and from brain of this and other species. In particular, the ATP concentration required by the 26S proteasome to reach maximal peptidase activity was approximately 40-fold lower than the one required for maximal proteolytic activity on polyubiquitinylated substrates. Moreover, plots of substrate concentration vs. velocity gave a saturation curve for the 26S proteasome only, which, for the trypsin-like and post-glutamyl peptide hydrolase activities fitted the Michaelis–Menten equation, whereas for the chymotrypsin-like activity indicated multibinding site kinetics with positive cooperativity (
n=2.32±0.38). As concerns the 20S proteasome, its electrophoretic pattern on native gel revealed a single protein band, a feature, to our knowledge, not yet described for the brain particle of any species.
Aims
In circulating lymphocytes of NIDDM patients pyruvate dehydrogenase (PDH), the major determinant in glucose consumption through oxidative pathways, is poorly active. The aim of this study is to ...examine whether sulphonylurea drug treatment revives PDH activity in circulating lymphocytes from NIDDM patients.
Methods
Twenty normal‐weight individuals with NIDDM were enrolled in this study. They had maintained their glycaemic levels close to normal by means of a restricted diet that had no longer been successful in the proceeding 2 months. The treatment protocol consisted in 160 mg gliclazide daily for 5 weeks. Twenty healthy subjects, matched for age, body mass index and gender, were enrolled as a control group. Patients, before and after treatment, as well as controls were tested for PDH activity in their circulating lymphocytes. Nine other untreated patients and nine healthy subjects, with the above mentioned characteristics, were recruited for the assay of PDH activity in their circulating lymphocytes before and after exposure, in vitro, to gliclazide, to insulin, and to gliclazide and insulin in combination.
Results
In gliclazide‐treated NIDDM patients, PDH activity in circulating lymphocytes recovered. In vitro, in circulating lymphocytes of untreated patients and controls insulin at 5 μU ml−1 was ineffective and highly effective, respectively, in raising enzyme activity; gliclazide at 10 ng ml−1 was ineffective on PDH in both groups, but in combination with insulin at 5 μU ml−1 in both groups PDH was as active as in cells of controls exposed to insulin only. In cells of controls, gliclazide alone at 25–50 ng ml−1 caused enzyme activation, whereas above 50 ng ml−1 it caused inhibition; in cells of patients below 50 ng ml−1 it had no effects, but at 50 ng ml−1 and above raised enzyme activity to the basal level of controls.
Conclusions
This study suggests that free gliclazide concentrations determine recovery of PDH activity in circulating lymphocytes of treated patients through drug‐mediated enhanced insulin control over PDH or through the drug alone.
BACKGROUND In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the ...transcriptome of mouse blastocysts. METHODS We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). CONCLUSIONS Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.
Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of ...primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.
Oxidation reactions on –OH groups at C-2 and C-3 positions of the uronic units of sodium alginate were performed with sodium periodate, and the influence of degree of oxidation on physical properties ...of the oxidized derivatives was analyzed. The aim of this work was to find new active functional groups on alginates, which is a polymer of interest as support in drug-controlled delivery systems. The molar mass decreases rapidly until an oxidation of 10
mol% and then remains nearly constant. Polymers with a degree of oxidation higher than 10
mol% were no more able to form gels with calcium ions. Both elastic modulus (
G′) and swelling degree (SD) of alginate gels decrease as the degree of oxidation increases. This behavior was attributed to the decreasing in the cooperative interactions between calcium ions and carboxylate groups due to a decrease in both the molar mass and the number of unreacted G units.
Mediterranean soil resources are heavily affected by water erosion. There is a need for anticipating the potential impact of land-use change at the catchment scale, considering a range of contrasted ...possible changes, in order to prepare a relevant adaptation strategy. While participatory scenario approaches are useful for unveiling the diversity of possible futures, their outputs generally take the form of narratives that may be difficult to relate to biophysical models used for simulating soil and water processes. We addressed this challenge by developing a methodology combining narrative scenarios and land-use change modelling at the catchment scale, interacting with local stakeholders who were involved throughout the research, in line with current Land System research. First, we built contrasted scenarios, each with a narrative story and quantitative assumptions for key variables. Then we drew up rules for translating narrative scenarios into spatially explicit maps based on expert groups and local knowledge, simulating the evolution of land use over time. Third, we evaluated and validated the final scenarios by conducting two workshops with scientists and local stakeholders. Finally, we assessed the impact of these scenarios on agricultural production and erosion control. This approach was implemented in the Tleta basin (Morocco), a 180 km2 rural watershed, 10 km from Tangiers and heavily affected by water erosion. As a result, we propose three contrasted spatially explicit land-use change scenarios by 2040. Our work highlights the interest of combining narrative storylines and land-use modelling when developing spatially explicit scenarios at the catchment scale, incorporating the local knowledge of stakeholders. The combination of Land System and ecosystem frameworks contributes to integrating the soil erosion issue in a more global perspective, and lays the foundation for building sustainable strategies for Mediterranean catchments affected by soil erosion.
•We combine narratives, land-use change modelling and participation at the catchment scale.•We elaborate rules for translating narratives into maps based on local knowledge.•We built three contrasted spatially explicit land-use change scenarios.•Land-use scenarios are used as concrete support for discussion with stakeholders.•We highlight potential services’ trade-offs between agro-ecological zones.
Reductive amination of chitosan (○) in presence of
o-phthaldialdehyde (■ and ●) inducing a large increase of the apparent molar mass.
In this paper, the reaction between
o-phthaldialdehyde and free
...NH
2 of chitosan is investigated; at a very low molar ratio between the two reactants (dialdehyde/
NH
2
∼
2.5
×
10
−4), an increase of the apparent molecular weight is obtained as evidenced from the rheological behaviour. Then, three non-ionic polysaccharides (galactomannan, maltodextrins, methylcellulose) are oxidised to 10% with sodium metaperiodate to obtain polyaldehydic derivatives able to react with free
NH
2 of chitosan after their direct dissolution into chitosan solution at a molar ratio monosaccharide units/
NH
2
∼
0.6. Stable swollen porous gels are obtained with an excellent yield in the presence of a reducing agent (NaBH
3CN) chosen to reduce the Schiff base; nearly no influence of the structure of the initial non-ionic polysaccharides is observed when the polysaccharides are oxidized in the same conditions. Different parameters for the reaction of oxidized methylcellulose (Me-ox) with chitosan are tested: influence of the degree of oxidation (up to 50%), and of the oxidised methylcellulose concentration. The larger is the degree of oxidation or the Me-ox concentration, the lower is the degree of swelling (i.e., the larger is the degree of chitosan cross-linkage). The swollen gels formed immediately after reaction are isolated and re-swell in aqueous acidic conditions, a good solvent of initial chitosan, to purify the gel and determine the yield of the reaction and the swelling degree. At the end, preliminary tests of biodegradability of these new gels are performed using specific enzymatic degradation with lysozyme and cellulase in the case of chitosan/Me-ox cogels chosen as example.
The influence of xanthan conformation on the physicochemical behaviour of their mixtures with galactomannan from
Schizolobium parahybae mannose:galactose ratio (M/G=3), was studied by viscoelastic ...measurements, differential scanning calorimetry (DSC) and chiroptical (circular dichroism) methods. The results suggested a more effective interaction of the galactomannan with disordered xanthan segments, which are more abundant in low salt concentrations but are still present in lower proportion at temperatures lower than the temperature of xanthan conformational transition (
T
m). The dependence of ellipticity with temperature in a circular dichroism (CD) spectra suggested an ordering of the xanthan chains induced by galactomannan at the temperature of gel formation (
T
g≈25°C), under conditions where xanthan alone exhibits a disordered conformation. The lower
T
g value found (≈25°C) compared with that (60°C) usually described in the literature is certainly related to the M/G ratio and the galactosyl unit distribution along the mannan main chain.
Proteasomes are multisubunit proteases involved in many cellular processes, including tumorigenesis and immune surveillance. In their catalytic core, the 20S proteasome, the beta1, beta2 and beta5 ...subunits show peptidylglutamyl peptide hydrolyzing (PGPH), trypsin-like and chymotrypsin-like activities, respectively. By IFN-gamma and TNFalpha stimulus, these subunits are replaced by their counterparts LMP2, MECL-1 and LMP7, defined inducible subunits, thus originating the immunoproteasome, and expression of the proteasome activator PA28 is enhanced. These modifications strengthen MHC-class I restricted peptide generation. The 20S proteasome has been detected immunohistochemically in formalin-fixed samples purified from fresh surgical specimens of 18 tumors (G20S) and from 8 samples of normal peritumoral tissue. The G20S, LMP2, MECL-1 and LMP7 increased in only 12 cases, along with unvaried trypsin-like and decreased PGPH and chymotrypsin-like activities; PA28 was unvaried in all 18 samples. The immunoproteasome alterations may represent an anomalous immunological attitude of glioblastomas.
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