Structural characterization of recombinant human myoglobin isoforms by (1)H and (129)Xe NMR and molecular dynamics simulations

Myoglobin (Mb), the main cytosolic oxygen storage/deliver protein, is also known to interact with different small ligands exerting other fundamental physiological roles. In Humans up to five different Mb isoforms are present. The two most expressed ones (>90%) differ only at the 54th position, K54 (Mb-I) and E54 (Mb-II) respectively. High-altitude populations are characterized by a higher Mb concentration in skeletal muscle, totally attributable to Mb-II, as well as a higher efficiency of locomotion, leading to the hypothesis of a cause-effect relationship with the evolutionary response to the high-altitude hypoxic environment. In this work, a first structural characterization of the two more expressed human Mb isoforms has been carried out. In particular, a detailed (1)H and (129)Xe NMR study was aimed to characterize the structure of the hydrophobic cavities around the heme group. Experimental results have been compared to those from MD simulations, i.e. volume fluctuations and occurrence. Electronic structure of the heme ring ground state resulted to be comparable for the two investigated isoforms, despite the single point mutation at position 54. However, the use of (129)Xe as a probe revealed small but significant modifications in the structure of internal cavities. MD simulations supported NMR results indicating interesting structural/dynamical differences in the average volume and occurrence of the main cavities lining Mb prosthetic group.     

The Diadenosine Homodinucleotide P18 Improves In Vitro Myelination in Experimental Charcot‐Marie‐Tooth Type 1A

Charcot‐Marie‐Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca²⁺ concentration ([Ca²⁺]_i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca²⁺ levels through down‐regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5′,5′′′‐P1, P2‐diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11‐overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]_i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild‐type (wt) cells. P18 increased [cAMP]_i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non‐phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]_i‐increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype. J. Cell. Biochem. 115: 161–167, 2014. © 2013 Wiley Periodicals, Inc.     

A Multilocus Genetic Study in a Cohort of Italian SLE Patients Confirms the Association with STAT4 Gene and Describes a New Association with HCP5 Gene

Background

Systemic lupus erythematosus (SLE) is an autoimmune disease with complex pathogenesis in which genes and environmental factors are involved. We aimed at analyzing previously identified loci associated with SLE or with other autoimmune and/or inflammatory disorders (STAT4, IL10, IL23R, IRAK1, PSORS1C1, HCP5, MIR146a, PTPN2, ERAP1, ATG16L1, IRGM) in a sample of Italian SLE patients in order to verify or confirm their possible involvement and relative contribution in the disease.

Materials and methods

Two hundred thirty-nine consecutive SLE patients and 278 matched healthy controls were enrolled. Study protocol included complete physical examination, and clinical and laboratory data collection. Nineteen polymorphisms were genotyped by allelic discrimination assays. A case-control association study and a genotype-phenotype correlation were performed.

Results

STAT4 was the most associated gene [P = 3×10⁻⁷, OR = 2.13 (95% CI: 1.59–2.85)]. IL10 confirmed its association with SLE [rs3024505: P = 0.02, OR = 1.52 (95% CI: 1.07–2.16)]. We describe a novel significant association between HCP5 locus and SLE susceptibility [rs3099844: P = 0.01, OR = 2.06 (95% CI: 1.18–3.6)]. The genotype/phenotype correlation analysis showed several associations including a higher risk to develop pericarditis with STAT4, and an association between HCP5 rs3099844 and anti-Ro/SSA antibodies.

Conclusions

STAT4 and IL10 confirm their association with SLE. We found that some SNPs in PSORS1C1, ATG16L1, IL23R, PTPN2 and MIR146a genes can determine particular disease phenotypes. HCP5 rs3099844 is associated with SLE and with anti-Ro/SSA. This polymorphism has been previously found associated with cardiac manifestations of SLE, a condition related with anti-Ro/SSA antibodies. Thus, our results may provide new insights into SLE pathogenesis.     

Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence.     

Muscarinic Receptor Subclasses in the Chick Embryo Retina: Influence of Corticosterone Treatment

The present study was performed on retinas of chick embryos receiving at day 8 of incubation an intracerebral injection of 0.02 microgram of corticosterone. We had previously shown with the use of [3H]quinuclidinylbenzilate [( 3H]QNB) that such treatment induced the appearance of two muscarinic binding sites in the treated retinas, whereas only one was detectable in the controls. In the present study we investigated muscarinic cholinergic receptor subclasses with agonist and antagonist binding. Agonist binding was studied by varying the concentrations of carbachol and acetylcholine (10(-9) M-10(-5) M) in the presence of a constant concentration (0.2 nM) of [3H]QNB. Two subpopulations of receptors were revealed, a high- and a low-affinity receptor, in both treated and control retinas. However, in the hormone-treated retinas, the two subpopulations significantly differed from the controls in their affinity and in their relative percentage among the total receptor population. Moreover, using pirenzepine, an antagonist known to have the capacity to distinguish between muscarinic cholinergic subclasses, two receptor subpopulations were found to be present in the hormone-treated retinas but a single one in the controls. It is suggested that hormone treatment can either induce the appearance of a new subclass of muscarinic cholinergic receptors or favor the maturation of a population of retinal cells having these receptors. Pirenzepine binding in retinas from intact embryos of 7, 9, and 11 days of incubation revealed one receptor subpopulation. Thus, these findings are more consistent with the hypothesis that corticosterone effects the target cells, either inducing changes in muscarinic receptor and/or modifying the receptor environment.     

Relationship between interphase AgNOR distribution and nucleolar size in cancer cells

Summary We have studied the relationship between interphase nucleolar organizer region (NOR) distribution and nucleolar size in cancer cells at light-microscopical level. Thirteen cases of formalin-fixed bladder cancer and fifteen cases of methacarn-fixed tumours of different origin were used. Nucleoli of the former cases were stained by Phloxine B and of the latter by Toluidine Blue. Selective visualization of interphase NORs was obtained by carrying out the one-step silver staining reaction for AgNOR proteins (Plotonet al., 1986). The area occupied by Phloxine B- or Toluidine Blue-stained nucleoli and interphase silver-stained NORs was measured by means of an automated image analyser. Both in bladder cancers and in the other tumour lesions nucleolar and interphase AgNOR areas were linearly related (r=0.95 and r=0.96, respectively,P<0.001). The close relationship between the area of nucleoli and that of silver-stained nucleolar structures was maintained even if the silver-staining procedure was prolonged beyond the optimal time length for selective interphase NOR staining. In the latter case, however, single interphase AgNORs were no longet visible within the nucleolar body which was, in fact, homogeneously stained. These data indicate that evaluation of the interphase AgNOR area has the same relevance, in tumour pathology, as whole nucleolar size measurement.     

EGF responsiveness of hepatocytes after partial hepatectomy

We investigate the effect of EGF on IP₃ production, PLCγ phosphorylation, calcium transients in rat hepatocytes isolated in quiescent liver (G₀ phase of cell cycle) and at 4 h (G₁ phase of cell cycle) and 24 h (M phase of cell cycle) after partial hepatectomy. Our results show that EGF does not utilize IP₃ and calcium as its signal transduction molecules when the hepatocytes are in vivo stimulated to entry in the cell cycle. In particular the growth factor does not phosphorylate PLCγ and induces a decrease in IP₃ content. These data suggest that EGF utilizes different signal transduction to send information from receptor to nucleus during PH with respect to the quiescent liver.     

Structure and absolute stereochemistry of vanillosmin,a guaianolide from Vanillosmopsis erythropappa

The structure and absolute stereochemistry of vanillosmin were established by chemical and spectral evidence and by comparison with O-acetyl-isophoto-α-santonic lactone and tetrahydroartabsin “C”.     

Effects of Cyclic Nucleotides and Calcium/Calmodulin on Protein Phosphorylation in the CNS of Hirudo medicinalis

Protein phosphorylation plays an important role in the regulation of neural functions. We have studied the phosphorylation of proteins in homogenates of segmental ganglia of the leech Hirudo medicinalis. We describe a number of proteins whose phosphorylation is dependent on calcium/calmodulin or cyclic nucleotides. Most of the proteins whose phosphorylation is increased in the presence of calcium seem to be substrates for cyclic nucleotide-dependent protein kinases. Only two of the phosphoproteins described appear to be specific substrates for calcium/calmodulin protein kinase(s), and at least six phosphoproteins appear to be specific substrates for cyclic nucleotide-dependent kinase(s). The leech nervous system, with large and identifiable neurons, provides a good tool for studies of neural functions, such as learning. The results are discussed in the context of the role of protein phosphorylation on learning processes.