Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Immune and Stress Responses Covary with Melanin-Based Coloration in the Barn Swallow

Eumelanin and pheomelanin are the main endogenous pigments in animals and melanin-based coloration has multiple functions. Melanization is associated with major life-history traits, including immune and stress response, possibly because of pleiotropic effects of genes that control melanogenesis. The net effects on pheo- versus eumelanization and other life-history traits may depend on the antagonistic effects of the genes that trigger the biosynthesis of either melanin form. Covariation between melanin-based pigmentation and fitness traits enforced by pleiotropic genes has major evolutionary implications particularly for socio-sexual communication. However, evidence from non-model organisms in the wild is limited to very few species. Here, we tested the hypothesis that melanin-based coloration of barn swallow (Hirundo rustica) throat and belly feathers covaries with acquired immunity and activation of the hypothalamic–pituitary–adrenal (HPA) axis, as gauged by corticosterone plasma levels. Individuals of both sexes with darker brownish belly feathers had weaker humoral immune response, while darker males had higher circulating corticosterone levels only when parental workload was experimentally reduced. Because color of belly feathers depends on both eu- and pheomelanin, and its darkness decreases with an increase in the concentration of eu- relative to pheomelanin, these results are consistent with our expectation that relatively more eu- than pheomelanized individuals have better immune response and smaller activation of the HPA-axis. Covariation of immune and stress response arose for belly but not throat feather color, suggesting that any function of color as a signal of individual quality or of alternative life-history strategies depends on plumage region.     

Fish oil supplementation for two generations increases insulin sensitivity in rats

We investigated the effect of fish oil supplementation for two consecutive generations on insulin sensitivity in rats. After the nursing period (21 days), female rats from the same prole were divided into two groups: (a) control group and (b) fish oil group. Female rats were supplemented with water (control) or fish oil at 1 g/kg body weight as a single bolus for 3 months. After this period, female rats were mated with male Wistar rats fed on a balanced chow diet (not supplemented). Female rats continued to receive supplementation throughout gestation and lactation periods. The same treatment was performed for the next two generations (G1 and G2). At 75 days of age, male offspring from G1 and G2 generations from both groups were used in the experiments. G1 rats did not present any difference with control rats. However, G2 rats presented reduction in glycemia and lipidemia and improvement in in vivo insulin sensitivity (model assessment of insulin resistance, insulin tolerance test) as well as in vitro insulin sensitivity in soleus muscle (glucose uptake and metabolism). This effect was associated with increased insulin-stimulated p38 MAP kinase phosphorylation and lower n-6/n-3 fatty acid ratio, but not with activation of proteins from insulin signaling (IR, IRS-1 and Akt). Global DNA methylation was decreased in liver but not in soleus muscle. These results suggest that long-term fish oil supplementation improves insulin sensitivity in association with increased insulin-stimulated p38 activation and decreased n-6:n-3 ratio in skeletal muscle and decreased global DNA methylation in liver.     

Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival,p53-dependent apoptosis and daunorubicin-induced cytotoxicity

Background

The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods

We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results

CK2α was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34⁺ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions

These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.

     

Historical Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.     

Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and Growth

It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis.     

Proteomic and Carbonylation Profile Analysis of Rat Skeletal Muscles following Acute Swimming Exercise

Previous studies by us and other groups characterized protein expression variation following long-term moderate training, whereas the effects of single bursts of exercise are less known. Making use of a proteomic approach, we investigated the effects of acute swimming exercise (ASE) on protein expression and carbonylation patterns in two hind limb muscles: the Extensor Digitorum Longus (EDL) and the Soleus, mostly composed of fast-twitch and slow-twitch fibres, respectively. Carbonylation is one of the most common oxidative modifications of proteins and a marker of oxidative stress. In fact, several studies suggest that physical activity and the consequent increase in oxygen consumption can lead to increase in reactive oxygen and nitrogen species (RONS) production, hence the interest in examining the impact of RONS on skeletal muscle proteins following ASE. Results indicate that protein expression is unaffected by ASE in both muscle types. Unexpectedly, the protein carbonylation level was reduced following ASE. In particular, the analysis found 31 and 5 spots, in Soleus and EDL muscles respectively, whose carbonylation is reduced after ASE. Lipid peroxidation levels in Soleus were markedly reduced as well. Most of the decarbonylated proteins are involved either in the regulation of muscle contractions or in the regulation of energy metabolism. A number of hypotheses may be advanced to account for such results, which will be addressed in future studies.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Ocular-Motor Profile and Effects of Memantine in a Familial Form of Adult Cerebellar Ataxia with Slow Saccades and Square Wave Saccadic Intrusions

Fixation instability due to saccadic intrusions is a feature of autosomal recessive spinocerebellar ataxias, and includes square wave intrusions (SWI) and macrosaccadic oscillations (MSO). A recent report suggested that the non-competitive antagonist of NMDA receptors, memantine, could decrease MSO and improve fixation in patients with spinocerebellar ataxia with saccadic intrusions (SCASI). We similarly tested two sisters, respectively of 58 and 60 years, with an unrecognized form of recessive, adult-onset cerebellar ataxia, peripheral neuropathy and slow saccades, who showed prominent SWI and also complained with difficulty in reading. We tested horizontal visually guided saccades (10°–18°) and three minutes of steady fixation in each patient and in thirty healthy controls. Both patients showed a significant reduction of peak and mean velocity compared with control subjects. Large SWI interrupting steady fixation were prominent during steady fixation and especially following visually guided saccades. Eye movements were recorded before and during the treatment with memantine, 20 mg/daily for 6 months. The treatment with memantine reduced both the magnitude and frequency of SWI (the former significantly), but did not modified neurological conditions or saccade parameters. Thus, our report suggests that memantine may have some general suppressive effect on saccadic intrusions, including both SWI and MSO, thereby restoring the capacity of reading and visual attention in these and in other recessive forms of ataxia, including Friedreich’s, in which saccadic intrusions are prominent.