Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis

Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease.     

Clastogenic and aneuploidizing effects of antiblastic busulphan revealed by kinetochore immunofluorescence in CHO cells.

We utilized, in CHO cells, the cytoplasm preservation technique to evaluate the micronucleus frequency at different busulphan concentrations, and the indirect immunofluorescence technique, using sera obtained from patients with scleroderma (CREST variant), to analyze if busulphan-induced micronuclei have kinetochores. Results show that this alkylating agent is capable of causing a significant increase of micronuclei in vitro, a great part (40%) of them having CREST-positive kinetochores. These findings confirm the clastogenic effect of busulphan and reveal a considerable capability of this agent to induce aneuploidy. These results are examined taking into account the high incidence of secondary neoplasias induced by chemotherapy with alkylating agents utilized against primary neoplasias.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

Monoclonal antibodies to human low density lipoprotein identify distinct areas on apolipoprotein B-100 relevant to the low density lipoprotein-receptor interaction.

We have characterized the epitopes for ten murine monoclonal antibodies (Mabs) to human low density lipoprotein (LDL) and studied their ability to interfere with the LDL-receptor interaction. The epitopes for the antibodies were defined by using the following approaches: 1) interaction with apoB-48; 2) interaction with apoB-100 thrombolytic fragments; and 3) interaction with beta-galactosidase-apoB fusion proteins spanning different areas of the apoB-100 sequence. The results obtained are consistent with the following map of epitopes: Mab 6E, amino acids (aa) 1-1297, Mabs 5A and 6B, aa 1480-1693, Mabs 2A, 7A, 3B, and 4B, aa 2152-2377, Mabs 8A and 9A, aa 2657-3248 and 3H, aa 4082-4306. Four Mabs (2A, 5A, 7A, and 9A) whose epitopes are located in three different areas of apoB, dramatically reduced (up to 95%) the LDL-receptor interaction on cultured human fibroblasts; Fab fragments were as effective as the whole antibodies. Mab 3H, on the other hand, increased LDL binding up to threefold. These findings are consistent with the hypothesis that several areas of apoB-100 are involved independently or in concert in modulating the apoprotein B conformation required for interaction with the LDL receptor.     

Analysis of mesophyll conductance in five understory herbaceous species

Mesophyll conductance (gm) has received over time much less attention than stomatal conductance (gs), although it affects leaf photosynthesis to about the same extent as stomatal conductance does. The objective of this study was to analyze the gm trend in five understory herbaceous species growing in a close-canopy forest in the north-west of Italy. In particular, three of analyzed species were monocots: Carex brizoides Lam., Carex pilosa Scop., and Oplismenus undulatifolius P. Beauv and the others dicots species: Circaea lutetiana L., and Pulmonaria officinalis Ced. The results showed, on one hand, the absence of correlation between gm and the considered environmental variables in the forest understory (i.e. air temperature, photosynthetic photon flux density and carbon dioxide concentration). Moreover, we carried out a principal component analysis considering all the analyzed morphological and physiological variables for the five species. The following correlation between the first component, related to the leaf mass per unit of leaf area and the leaf tissue density, and gm seem to suggest a key role of the leaf structural features in determining gm variations across the five species.     

Prime Ricerche sulla Fisionomia Enzimatica Caratteristica della Fotosintesi

Summary

The rate of oxidation of glucose-6-phosphate, ribose-5-phosphate, fructose-1,6- phosphate, iso-citrate and malate in extracts from green and etiolated pea leaves was determined, using the triphenyltetrazolium technique.

Glucose-6-phosphate, ribose-5-phosphate, and fructose-1,6-phosphate, in the presence of added TPN, where oxidized at a rate about twice higher in the extracts from green than in the extracts from etiolated leaves. Iso-citrate, in the presence of TPN, fructose-1,6-phosphate, in the presence of DPN, and malate, in the presence of either TPN or DPN, were oxidized at about the same rate in the two types of extracts.

These data seem to indicate a preferential synthesis of enzymes involved in the metabolic cycle of phosphorylated sugars during the transition of the leaf from the etiolated to the photosynthetising physiognomy. They seem also favourable to the view assigning to this metabolic system a primary importance in the anabolic pathway of photosynthesis.     

Mechanistic insight into the catechol oxidase activity by a biomimetic dinuclear copper complex

The biomimetic catalytic oxidation of 3,5-di-tert-butylcatechol by the dicopper(II) complex of the ligand ,-bis{bis[1-(1-methyl-2-benzimidazolyl)methyl]amino}-m-xylene in the presence of dioxygen has been investigated as a function of temperature and pH in a mixed aqueous/organic solvent. The catalytic cycle occurs in two steps, the first step being faster than the second step. In the first step, one molecule of catechol is oxidized by the dicopper(II) complex, and the copper(II) centers are reduced. From the pH dependence, it is deduced that the active species of the process is the monohydroxo form of the dinuclear complex. In the second step, the second molecule of catechol is oxidized by the dicopper(I)-dioxygen complex formed upon oxygenation of the reduced complex. In both cases, catechol oxidation is an inner-sphere electron transfer process involving binding of the catechol to the active species. The binary catechol-dicopper(II) complex formed in the first step could be characterized at very low temperature (–90 °C), where substrate oxidation is blocked. On the contrary, the ternary complex of dicopper(I)-O₂-catechol relevant to the second step does not accumulate in solution and could not be characterized, even at low temperature. The investigation of the biphasic kinetics of the catalytic reaction over a range of temperatures allowed the thermodynamic (H° and S°) and activation parameters (H and S) connected with the key steps of the catecholase process to be obtained.     

Relationship between two proprioceptive measures and stiffness at the ankle.

Previous research has investigated the role of proprioception and stiffness in the control of joint stability. However, to date, no research has been done on the relationship between proprioception and stiffness. Therefore, the purpose of this study was to determine the relationship between force sense, joint reposition sense, and stiffness at the ankle. A heterogeneous sample was obtained for this study; 20 of the 40 participants had a history of ankle sprains, and 13 of the 20 had been diagnosed by a physician (two mild ankle sprains, seven moderate sprains, four severe sprains). All subjects were asymptomatic and active at the time of the study. Active joint reposition sense was measured using a custom-built ankle goniometer, force sense was measured unilaterally and contralaterally with a load cell, and ankle muscle stiffness was measured via transient oscillation using a custom-built inversion-eversion cradle. We found no significant correlations between stiffness and joint reposition sense, with values of r ranging from 0.01 to 0.21. Significant correlations were found between stiffness and force sense. Specifically, contralateral force sense reproduction was significantly correlated to stiffness in the injured or "involved" ankle (r's ranging from 0.47 to 0.65; P< or =0.008). Whether the decreased ability to appropriately sense force (increased error) sends information to the central nervous system to increase muscle stiffness in response to an unexpected loss of stability, or whether these two phenomena function independently and both change concurrently as a result of injury to the system requires further investigation.