Fecal Short Chain Fatty Acids and Dietary Intake in Italian Women With Restrictive Anorexia Nervosa: A Pilot Study

Nutritional disorders such as Anorexia Nervosa (AN) can shape the composition of gut microbiota and its metabolites such as short chain fatty acid (SCFA). This study aims to compare fecal SCFA along with dietary intake of women with restrictive AN (r-AN = 10) and those of sex-matched lean controls (C = 8). The main fecal short chain fatty acids (SCFA) were assessed by gas chromatography equipped with a flame ionization detector. All participants completed 7-day food record and underwent indirect calorimetry for measuring resting energy expenditure (REE). Butyrate and propionate fecal concentrations were significantly reduced in r-AN patients compared to controls. The intake of carbohydrate and fat was significantly lower in r-AN patients than controls as well as energy intake and REE; whereas the amount of protein and fiber did not differ between groups. These preliminary results showed that r-AN patients had a reduced excretion of fecal SCFA, likely as a mechanism to compensate for the lower energy and carbohydrate intake observed between groups. Therefore, further studies need to be performed in patients with AN to explore the link between nutritional disorders, gut microbiota and its metabolites

Role for Non-Proteolytic Control of M-phase Promoting Factor Activity at M-phase Exit

M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation

Early diagnosis of multiple sclerosis

Multiple sclerosis (MS) is characterized by a chronic inflammation, demyelination and gliosis resulting in damage of the central nervous system (CNS). No definitive diagnostic tests for multiple sclerosis is currently available. Using the CSF from MS patients on cultures of differentiating oligodendrocytes (MO3.13), we have found that the expression of a specific Marker (Marker C) is significantly reduced compared with control groups. Marker-C is a stress marker. We do not disclose the identity of Marker-C because it is under patentin process as a marker of early multiple sclerosis. Moreover, we have found in CSF from MS patients molecules that act on redox pathways and interfere with hydrogen peroxide induced oligodendrocyte maturation. In differentiated MO3.13 cells, treated with CSF from MS patients, there is also, a significant increase of other 2 markers (Marker-A and Marker-B), which are negative differentiation markers, compared to cells treated with CSF from control group. These results indicate that CSF from MS patients contain specific molecule(s) that inhibit the oligodendrocyte differentiation by increasing the expression of the Marker-A and -B. Combining the variation of the stress marker, Marker-C, with the negative differentiation markers, Marker-A and -B, we have developed a mathematical MS index (AxB/C). We estimate that the test can detect a MS patient with a +/-5% error (false negatives). The identification of specific markers of oxidative stress and/or negative differentiation (markers A, B and C) modulated in M03.13 cells after MS CSF stimulation, may represent an indirect measurement of a specific cell receptor activation. This assay can be used as guide to develop a new diagnostic method for an early diagnosis of MS. Our test provides an opportunity to measure at an early stagethe activation of a specific signal transduction pathway resulting in the block of differentiation of oligodendrocytes. We conclude that in the cerebrospinal fluid (CSF)derived from patients with multiple sclerosis there is a molecule(s) that acts on oxidative stress pathways and that inhibits oligodendrocyte differentiation, altering the normal re-myelinization process

Figure 5

<p>Wee1 and cdk1 physically interact in M-phase. (<i>A</i>) Histone H1 kinase activity in cdk1 Ips from samples of CSF-arrested extract (M), interphase extract (I; 40 min after CaCl₂ and CHX additions) and from samples taken at the indicated time points after sea urchin ΔB (100nM) addition to an interphase extract. Left panel, phosphorylated histone H1 autoradiograph (P-HH1). Right panel, quantisation of cdk1 activity from three independent experiments. (<i>B</i>) Cdk1 activity, cdk1-phospho-tyr15 and cdk1 content in cdk1 Ips from extract samples taken at the indicated time points after ΔB addition. (<i>C, D</i>) Wee1 and cdk1 were immunoprecipitated from samples of CSF-arrested (M), interphase (I) extracts and interphase extract samples from the time of ΔB addition. Left panels, wee1 and wee1-associated cdk1 in wee1 Ips. Right, histone H1 kinase activity in cdk1 Ips. In (<i>D</i>) the lower part of the wee1 Ips immunoblot was first probed for cdk1-phospho-tyr15 and subsequently for cdk1. (<i>E, F</i>) Full-length, [³⁵S]labelled, Xenopus cyclin B1 was added to two independent interphase extracts along with ΔB. The extracts were spilt into two portions and DMSO, as control, or roscovitine (10 µM) were added after 33 min incubation, samples were, then, taken at the indicated time points after ΔB addition. Shown are autoradiographs and quantisations of percent remaining [³⁵S]labelled, full-length cyclin B1.</p

Figure 4

<p>MPF hampers Cdc20-APC/C interaction through Cdc20 phopshorylation. (<i>A</i>) Cyclin B stability after CaCl₂ addition in [³⁵S]labelled CSF-arrested extract portions treated with buffer, as control, recombinant cyclin B1-cdk1wt or cyclin B1-cdk1AF complexes. To portions of the cyclin B1-cdk1AF-treated extract, either roscovitine (2 µM; 1/30 extract volume), or DMSO (1/30 extract volume) as control, were added 1 min after CaCl₂. (<i>B</i>) Total Cdc27 and Cdc27-bound Cdc20 (IpCdc27/IbCdc20) from CSF-arrested samples treated with recombinant cyclin B1-cdk1wt and cyclin B1-cdk1AF complexes at the indicated time points after CaCl₂ addition (the time 0 sample received no CaCl₂). [³⁵S]labelled Cdc20 wild type (wt) and a 7 phosphorylation sites mutant version (7A) were produced in reticulocyte lysates (lanes 1, 2). Labelled proteins were incubated with portions of a CHX-treated CSF-arrested extract for 30 min (lanes 3, 4). Cdc27 was, then, immunoprecipitated (Cdc 27 Ip) and the amount of bound wt (lane 7) and 7A (lane 8) Cdc20 detected by autoradiography. Lanes 5, 6, mock Ips (Mk Ip). (<i>C</i>) Portions of a [³⁵S]labelled CSF-arrested extract were incubated for 40 min with mock (contr.), cdc20 wt or cdc20 7A programmed reticulocyte lysates in the presence of CHX. (<i>D</i>) The cdc20 wt- and cdc20 7A-treated portions where further incubated for 20 min with cyclin B1-cdk1AF. Then, aliquots were taken at the indicated time points after CaCl₂ addition. Shown is an autoradiograph of [³⁵S]labelled extracts proteins (the position of cyclin B is indicated). (<i>E</i>) Quantisation of remaining cyclin, expressed as percent, from cyclin B1-cdk1AF-treated extract portions in the presence of cdc20 wt (open squares) or cdc20 7A (filled squares). Error bars refer to variability within three independent experiments.</p

Figure 2

<p>Cyclin B1 abundance and MPF activity in CSF-arrested extracts. (<i>A</i>) Cyclin B, cdk1-phospho-tyr15, cdk1, cdc25C-phospho-ser-287 and cdc25C contents from total extract samples of a CSF-arrested extract, pre-incubated with [³⁵S]methionine, at the indicated time points after CaCl₂ addition. (<i>B</i>) Left panels, cyclin B1, cyclin B1-associated kinase activity, cdk1-phospho-tyr15 and cdk1 contents in cyclin B1 Ips from CSF-arrested extract samples at the indicated time points after CaCl₂ addition. Right, quantisation of cyclin B1 content (open squares), cyclin B1-associated kinase activity (filled squares) from cyclin B1 Ips.</p