Relationship between cAMP and Ca2+ fluxes in human platelet membranes

The effect of cAMP (which involved a 23 kDa protein phosphorylation) has been studied on the Ca2+ uptake and Ca2+ release from a human platelet membrane vesicle fraction. It was tested in the presence of the catalytic subunit of the cAMP-dependent protein kinase (C Sub). The addition of C Sub increased the steady state level of the Ca2+ uptake into the membrane vesicles. The effect was enhanced when tested in the absence of Ca2+ precipitating agent. The response was proportional to the dose of C Sub. Moreover, the effect varied with the Ca2+ concentration. The effect of C Sub has been tested on the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. A phosphorylated state of the 23 kDa protein appeared to be necessary. Indeed, a phosphorylation inhibition prevented the IP3 effect and the addition of C Sub increased the percentage of released Ca2+ (without modification of the time course). However, the C Sub dose-dependent response was not linear. The effect of cAMP on the two functions (Ca2+ uptake and Ca2+ release) appears to be different. Therefore, these results led us to suggest a more complex role of cAMP in the regulation of platelet Ca2+ concentration.     

Analysis of products involved in primary and secondary allogenic proliferation in man

An informative family, in which parents shared HLA-Dw and Ia-like DRw (Ly-Li) antigens, was used to produce PLTs between members either phenoidentical for both Dw and DRw determinants or incompatible for Dw specificities only. These PLTs were restimulated by members of the family: two PLTs, although in DRw identity, reacted against members of the family bearing one maternal (c) and/or one paternal (a) haplotype. A third PLT also developed in DRw identity reacted with members bearing the other maternal (d) haplotype. Population studies with one of these PLTs did not show any correlation with Dw or DRw specificities. Family studies are in keeping, but do not demonstrate an HLA linkage. The data suggest that, along with the stimulating products (PL^A) identical or closely related to the DRw determinants, other stimulating products (PL^B), also probably HLA-linked, exist. Furthermore, one of the PLTs was produced without a primary MLR.     

Proteomics advances for precision therapy in ovarian cancer

ABSTRACT

Introduction: Due to the relatively low mutation rate and high frequency of copy number variation, finding actionable genetic drivers of high-grade serous carcinoma (HGSC) is a challenging task. Furthermore, emerging studies show that genetic alterations are frequently poorly represented at the protein level adding a layer of complexity. With improvements in large-scale proteomic technologies, proteomics studies have the potential to provide robust analysis of the pathways driving high HGSC behavior.

Areas covered: This review summarizes recent large-scale proteomics findings across adequately sized ovarian cancer sample sets. Key words combined with ‘ovarian cancer’ including ‘proteomics’, ‘proteogenomic’, ‘reverse-phase protein array’, ‘mass spectrometry’, and ‘adaptive response’, were used to search PubMed.

Expert opinion: Proteomics analysis of HGSC as well as their adaptive responses to therapy can uncover new therapeutic liabilities, which can reduce the emergence of drug resistance and potentially improve patient outcomes. There is a pressing need to better understand how the genomic and epigenomic heterogeneity intrinsic to ovarian cancer is reflected at the protein level and how this information could be used to improve patient outcomes.     

Evidence of Coat Color Variation Sheds New Light on Ancient Canids

We have used a paleogenetics approach to investigate the genetic landscape of coat color variation in ancient Eurasian dog and wolf populations. We amplified DNA fragments of two genes controlling coat color, Mc1r (Melanocortin 1 Receptor) and CBD103 (canine-β-defensin), in respectively 15 and 19 ancient canids (dogs and wolf morphotypes) from 14 different archeological sites, throughout Asia and Europe spanning from ca. 12 000 B.P. (end of Upper Palaeolithic) to ca. 4000 B.P. (Bronze Age). We provide evidence of a new variant (R301C) of the Melanocortin 1 receptor (Mc1r) and highlight the presence of the beta-defensin melanistic mutation (CDB103-K locus) on ancient DNA from dog-and wolf-morphotype specimens. We show that the dominant K^B allele (CBD103), which causes melanism, and R301C (Mc1r), the variant that may cause light hair color, are present as early as the beginning of the Holocene, over 10 000 years ago. These results underline the genetic diversity of prehistoric dogs. This diversity may have partly stemmed not only from the wolf gene pool captured by domestication but also from mutations very likely linked to the relaxation of natural selection pressure occurring in-line with this process.     

Heme oxygenase-1 induction by nitric oxide in RAW 264.7 macrophages is upregulated by a cyclo-oxygenase-2 inhibitor

Unstimulated RAW 264.7 macrophages express negligible heme oxygenase-1 (HO-1) protein but incubation with the nitric oxide (NO) donor spermine nonoate (SPNO) induced HO-1 and weakly cyclo-oxygenase-2 (COX-2) protein. This effect was potentiated by coincubation with the COX-2 selective inhibitor, SC58125. Cells incubated with SPNO showed a strong increase in HO-1 mRNA levels after 4 h with a significant potentiation in the presence of SC58125, which did not modify HO-1 mRNA stability. The induction of HO-1 by NO and its potentiation by anti-inflammatory agents may play a role in inflammatory and immune responses.     

CD4-dependent generation of dominant transplantation tolerance induced by simultaneous perturbation of CD154 and LFA-1 pathways

CD154 and LFA-1 (CD11a) represent conceptually distinct pathways of receptor/ligand interactions (costimulation and adhesion/homing, respectively) that have been effectively targeted to induce long-term allograft acceptance and tolerance. In the current study, we determined the relative efficacy and nature of tolerance induced by mAbs specific for these pathways. In vitro analysis indicated that simultaneous targeting of CD154 and LFA-1 resulted in profound inhibition of alloreactivity, suggesting that combined anti-CD154/anti-LFA-1 therapy could be highly effective in vivo. Thus, we evaluated combining mAb therapies targeting CD154 and LFA-1 for inducing transplantation tolerance to pancreatic islet allografts. Monotherapy with either anti-CD154 or anti-LFA-1 was partially effective for inducing long-term allograft survival, whereas the combination resulted in uniform allograft acceptance in high-responder C57BL/6 recipients. This combined therapy was not lymphocyte depleting and did not require the long-term deletion of donor-reactive T lymphocytes to maintain allograft survival. Importantly, combined anti-CD154/anti-LFA therapy uniquely resulted in "dominant" transplantation tolerance. Therefore, simultaneous perturbation of CD154 and LFA-1 molecules can result in profound tolerance induction not accomplished through individual monotherapy approaches. Furthermore, results show that such regulatory tolerance can coexist with the presence of robust anti-donor reactivity, suggesting that active tolerance does not require a corresponding deletion of donor-reactive T cells. Interestingly, although the induction of this regulatory state was highly CD4 dependent, the adoptive transfer of tolerance was less CD4 dependent in vivo.     

Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA-binding protein HuR increases the half-life of some messenger RNAs that contain adenylate/uridylate-rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes. This work provides the first evidence for a physiological function of HuR, and highlights its involvement in spermatogenesis.     

Loss of α1β1 Soluble Guanylate Cyclase,the Major Nitric Oxide Receptor,Leads to Moyamoya and Achalasia

Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal “moyamoya” vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya.