Aims
Cytoplasmic accumulation of the nuclear protein transactive response DNA‐binding protein 43 (TDP‐43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis ...(ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP‐43‐coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP‐43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS‐linked mutant genes.
Methods
TDP‐43 subcellular localization was analysed with western immunoblotting and immunocytofluorescence in CLM of healthy controls (n = 10), patients with mutant TARDBP (n = 4, 1 homozygous), valosin‐containing protein (VCP; n = 2), fused in sarcoma/translocated in liposarcoma (FUS; n = 2), Cu/Zn superoxide dismutase 1 (SOD1; n = 6), chromosome 9 open reading frame 72 (C9ORF72; n = 4), without mutations (n = 5) and neurologically unaffected subjects with mutant TARDBP (n = 2).
Results
TDP‐43 cytoplasmic accumulation was found (P < 0.05 vs. controls) in CLM of patients with mutant TARDBP or VCP, but not FUS, in line with TDP‐43 subcellular localization described for motor neurons of corresponding groups. Accumulation also characterized CLM of the healthy individuals with mutant TARDBP and of some patients with mutant SOD1 or C9ORF72. In 5 patients, belonging to categories described to carry TDP‐43 mislocalization in motor neurons (3 C9ORF72, 1 TARDBP and 1 without mutations), TDP‐43 cytoplasmic accumulation was not detected in CLM or in lymphocytes but was in monocytes.
Conclusions
In ALS forms characterized by TDP‐43 mislocalization in motor neurons, monocytes display this alteration, even when not manifest in CLM. Monocytes may be used to support diagnosis, as well as to identify subjects at risk, of ALS and to develop/monitor targeted treatments.
Altered sub‐cellular localisation of TDP‐43 in monocytes may represent a new diagnostic tool and investigation of monocytes may improve our understanding of disease mechanisms in ALS.
Proteasomes constitute the degradative machinery of the ubiquitin/adenosine triphosphate-dependent proteolytic pathway, which is involved in many cell functions, including immune response and ...apoptosis, and in HIV maturation and infectivity.
To examine whether proteasomes are targeted by antiretroviral agents.
Chymotrypsin-like, trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of purified human 26S and 20S proteasomes, the latter depleted or enriched in 11S regulator, were assayed after incubation with indinavir, lamivudine and zidovudine at 1-80 microM alone and in combination. To assess the drug effects on cellular functions regulated by proteasomes, the accumulation of ubiquitin-tagged proteins, the processing of the nuclear factor kappa B precursor p105, and the degradation of the inhibitor of nuclear factor kappa B, isoform alpha (IkappaBalpha) were evaluated by Western immunoblotting in Jurkat cells after incubation for 6 h with the drugs above.
Trypsin-like and mostly chymotrypsin-like activities of purified 26S proteasome were inhibited by each drug from 10 to 80 microM, more by double combinations and mostly by the triple combination. The peptidyl-glutamyl-peptide hydrolysing activity of the 26S proteasome and the three peptidase activities of the 20S proteasome, depleted or enriched in 11S regulator, were unaffected. The accumulation of ubiquitin-tagged proteins, reduced IkappaBalpha degradation and p105 processing were appreciable in intact cells with the triple drug combination.
The human 26S proteasome is a target of antiretroviral agents. This suggests that the antiviral action and some clinical and immunological benefits of combined antiretroviral therapy rely not only on its known effects on viral enzymes, but also on host cell components.
Chitin is the second most important natural polymer in the world. The main sources exploited are two marine crustaceans, shrimp and crabs. Our objective is to appraise the state of the art concerning ...this polysaccharide: its morphology in the native solid state, methods of identification and characterization and chemical modifications, as well as the difficulties in utilizing and processing it for selected applications. We note the important work of P. Austin, S. Tokura and S. Hirano, who have contributed to the applications development of chitin, especially in fiber form. Then, we discuss chitosan, the most important derivative of chitin, outlining the best techniques to characterize it and the main problems encountered in its utilization. Chitosan, which is soluble in acidic aqueous media, is used in many applications (food, cosmetics, biomedical and pharmaceutical applications). We briefly describe the chemical modifications of chitosan—an area in which a variety of syntheses have been proposed tentatively, but are not yet developed on an industrial scale. This review emphasizes recent papers on the high value-added applications of these materials in medicine and cosmetics.
In most tissues expressing MHC class I molecules, proteasomes incorporating IFN-γ-inducible subunits, defined immuno-proteasomes, exist together with constitutive proteasomes. In physiological ...conditions, the central nervous system expresses neither MHC class I molecules nor TAP1 and TAP2 transporters but besides being constitutive, it is unknown whether immuno-proteasomes are also present in this tissue. We present evidence that in human brain, the two types of proteasome exist suggesting that under physiological conditions, the mechanisms regulating expression of IFN-γ-inducible subunits as well as of MHC class I molecules and TAP1 and TAP2 transporters in nervous tissue, are not entirely coordinated.
Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are ...used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.
During angiogenesis, a combined action between newly secreted extracellular matrix proteins and the repertoire of integrins expressed by endothelial cells contributes in the regulation of their ...biological functions. Extracellular matrix-engaged integrins influence tyrosine kinase receptors, thus promoting a regulatory cross-talk between adhesive and soluble stimuli. For instance, vitronectin has been reported to positively regulate VEGFR-2. Here, we show that collagen I downregulates VEGF-A-mediated VEGFR-2 activation. This activity requires the tyrosine phosphatase SHP2, which is recruited to the activated VEGFR-2 when cells are plated on collagen I, but not on vitronectin. Constitutive expression of SHP2(C459S) mutant inhibits the negative role of collagen I on VEGFR-2 phosphorylation. VEGFR-2 undergoes internalisation, which is associated with dynamin II phosphorylation. Expression of SHP2(C459S) impairs receptor internalisation suggesting that SHP2-dependent dephosphorylation regulates this process. These findings demonstrate that collagen I in provisional extracellular matrix surrounding nascent capillaries triggers a signaling pathway that negatively regulates angiogenesis.
In circulating lymphomonocytes (CLM) of patients with Type 2 diabetes (DM2) pyruvate dehydrogenase (PDH), the major determinant of glucose oxidative breakdown, is affected by a cohort of alterations ...reflecting impaired insulin-stimulated glucose utilization. The cohort is also expressed, although incompletely, in 40% of healthy young subjects with a DM2-family history (FH). Pregnancy restrains glucose utilization in maternal peripheral tissues to satisfy fetal requirements. Here we explore whether pregnant women develop the PDH alterations and, if so, whether there are differences between women with and without FH (FH+, FH−). Ten FH+ and 10 FH — were evaluated during pregnancy (12–14, 24–26, and 37–39 weeks) and 1 yr after (follow-up) for fasting plasma glucose and insulin as well as body mass index (BMI), and for the PDH alterations. Twenty FH — and 20 FH+ non-pregnant women served as controls. All FH+ and FH — controls exhibited normal clinical parameters and 8 FH+ had an incomplete cohort of PDH alterations. In FH — and FH+ pregnant women at 12–14 weeks clinical parameters were normal; from 24–26 weeks, with unvaried glucose, insulin and BMI rose more in FH — and only in the latter recovered the 12–14 weeks values at follow-up. In all FH−, the cohort of PDH alterations was incomplete at 24–26 weeks, complete at 37–39 weeks, and absent at follow-up but complete from 12–14 weeks including follow-up in all FH+. In FH−, the cohort is an acquired trait restricted to pregnancy signaling transiently reduced insulin-stimulated glucose utilization; in FH+, instead, it unveils the existence of an inherited DM2-related background these women all have, that is awakened by pregnancy and as such lastingly impairs insulin-stimulated glucose utilization.
Pyruvate dehydrogenase (PDH) has low activity in the circulating lymphocytes (CL) of obese adolescents and adults. In vitro, it is unresponsive to insulin at 5 micro-units/ml and is activated at 50 ...micro-units/ml, in contrast with activation and inhibition respectively at these concentrations in CL from controls. These changes are seen as being indicative of a molecular disorder underlying insulin resistance. The aims of the present study were to determine whether a substantial enhancement of blood insulin levels restores the PDH activity in CL from obese adolescents and abolishes the in vitro alterations, and whether PDH activity and indices of insulin resistance are correlated. Six obese adolescents and six normal-weight controls underwent a 4 h frequently sampled intravenous glucose test with minimal model analysis, to bring about a sharp rise in blood insulin and provide a reliable index of insulin sensitivity (S(I)). PDH activity was evaluated in CL obtained from blood samples at set times before and after their exposure to insulin in vitro. Insulin levels rose in all subjects in the first 10 min, although to a much greater extent in the obese group, and then decreased until the end of the test (240 min; t(240)). PDH activity in CL paralleled the insulin pattern in the control subjects, whereas in the obese subjects it was below normal 3 min before the start of the test (t(-3)), but rose significantly throughout the test. PDH responses in vitro to insulin in CL taken from the control subjects at t(-3) and t(240) and in CL taken from the obese subjects at t(-3) were as reported above, but were normal (i.e. the same as in control CL) in CL taken from the obese subjects at t(240). Baseline PDH activity was inversely correlated with body mass index and with fasting insulin, and directly correlated with S(I). These results show that a brief and sharp enhancement of blood insulin overcomes derangements in PDH that reflect systemic insulin resistance in obese adolescents.
Background
Glucocorticoid administration induces alteration of glucose tolerance, and impairment of glucose oxidation may contribute to glucocorticoid‐induced derangement of glucose metabolism. We ...investigated glucose tolerance following methylprednisolone administration in humans. In the same model, we evaluated pyruvate dehydrogenase (PDH), the rate limiting enzyme of glucose oxidation, in peripheral blood mononuclear cells.
Materials and methods
Methylprednisolone (2 × 40 mg, iv, one dose every 12 h) was administered to six healthy volunteers. Glucose tolerance was evaluated through an oral glucose tolerance test (oGTT, 75 g glucose) at least a week before and after drug administration (2 and 24 h post‐drug). To assess modifications of lipid metabolism circulating free fatty acids (FFA) and glycerol were measured, during fasting and oGTT. The active form of PDH (PDHa) was evaluated in peripheral blood mononuclear cells, both as ex vivo activity and as in vitro response to insulin (30pmol l−1).
Results
Methylprednisolone induced an alteration of glucose tolerance 2 h after its administration. Such alteration was completely reversed at 24 h. Alteration of glucose tolerance was accompanied by decreased ex vivo PDHa activity. PDH responsiveness to insulin in vitro was also impaired. Circulating FFA were unmodified, but decreased glycerol levels suggested a slight inhibition of lipolysis.
Conclusions
Acute methylprednisolone administration in humans induced a transient decrease of glucose tolerance 2 h after drug administration, accompanied by hyperinsulinaemia, inhibition of ex vivo PDH activity and its response to insulin in vitro. These alterations were completely abolished at 24 h, suggesting that methylprednisolone can be safely administered acutely. Furthermore, methylprednisolone induced only minor modifications of circulating FFA and glycerol, indicating minimal impact on lipid metabolism.
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