REGULATION OF MEMBRANE BAND 3 TYR-PHOSPHORYLATION BY PROTEOLYSIS OF P72 AND POSSIBLE INVOLVEMENT IN SENESCENCE PROCESS

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation (Tyr-P) of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa (p72Syk) to the 36 kDa (p36Syk) isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32P uptake into the cytoplasmic domain of band 3 after addition of p72Syk or p36Syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36Syk on p72Syk-mediated phosphorylation and the effect of addition of a protease inhibitor (leupeptin) on p72Syk-mediated phosphorylation were studied by autoradiographic visualization of 32P uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36Syk had a higher band 3 tyrosine phosphorylating activity compared with p72Syk. Pre-phosphorylation with p36Syk or p72Syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72Syk but not p36Syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72Syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton

Olfactory neuroepithelium alterations and cognitive correlates in schizophrenia

BACKGROUND: Few studies have investigated alterations of olfactory neuroepithelium (ONE) as a biomarker of schizophrenia, and none its association with cognitive functioning. METHOD: Fresh ONE cells from twelve patients with schizophrenia and thirteen healthy controls were collected by nasal brushing, cultured in proper media and passed twelve times. Markers of cell proliferation (BrdU incorporation, Cyclin-D1 and p21 protein level) were quantified.Cognitive function was measured using Brief Neuropsychological Examination-2. PRIMARY OUTCOME: proliferation of ONE cells from schizophrenic patients at passage 3. Secondary outcome: association between alteration of cell proliferation and cognitive function. RESULTS: Fresh ONE cells from patients showed a faster cell proliferation than those from healthy controls at passage 3. An opposite trend was observed at passage 9, ONE cells of patients with schizophrenia showing slower cell proliferation as compared to healthy controls. In schizophrenia, overall cognitive function (Spearman's rho -0.657, p\u202f<\u202f0.01), verbal memory - immediate recall, with interference at 10\u202fs and 30\u202fs (Spearman's rho from -0.676 to 0.697, all p\u202f<\u202f0.01) were inversely associated with cell proliferation at passage 3. CONCLUSION: Fresh ONE cells collected by nasal brushing might eventually represent a tool for diagnosing schizophrenia based upon markers of cell proliferation, which can be easily implemented as single-layer culture. Cell proliferation at passage 3 can be regarded as a promising proxy of cognitive functioning in schizophrenia. Future studies should replicate these findings, and may assess whether ONE alterations are there before onset of psychosis, serving as an early sign in patients with at risk mental state

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration

Epidemiology and outcome of Clostridium difficile infections in patients hospitalized in Internal Medicine: findings from the nationwide FADOI-PRACTICE study.

BACKGROUND: Clostridium difficile (CD) is a leading cause of diarrhoea among hospitalized patients. The objective of this study was to evaluate the rate, the optimal diagnostic work-up, and outcome of CD infections (CDI) in Internal Medicine (IM) wards in Italy. METHODS: PRACTICE is an observational prospective study, involving 40 IM Units and evaluating all consecutive patients hospitalized during a 4-month period. CDI were defined in case of diarrhoea when both enzyme immunoassay for GDH, and test for A/B toxin were positive. Patients with CDI were followed-up for recurrences for 4 weeks after the end of therapy. RESULTS: Among the 10,780 patients observed, 103 (0.96 %) showed CDI, at admission or during hospitalization. A positive history for CD, antibiotics in the previous 4 weeks, recent hospitalization, female gender and age were significantly associated with CDI (multivariable analysis). In-hospital mortality was 16.5 % in CD group vs 6.7 % in No-CD group (p\u2009&lt;\u20090.001), whereas median length of hospital stay was 16 (IQR\u2009=\u200913) vs 8 (IQR\u2009=\u20098) days (p\u2009&lt;\u20090.001) among patients with or without CDI, respectively. Rate of CD recurrences was 14.6 %. As a post-hoc evaluation, 23 out of 34 GDH+/Tox- samples were toxin positive, when analysed by molecular method (a real-time PCR assay). The overall CD incidence rate was 5.3/10,000 patient-days. CONCLUSIONS: Our results confirm the severity of CDI in medical wards, showing high in-hospital mortality, prolonged hospitalization and frequent short-term recurrences. Further, our survey supports a 2-3 step algorithm for CD diagnosis: EIA for detecting GDH, A and B toxin, followed by a molecular method in case of toxin-negative samples

The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins

Studies on the actin-binding protein HS1 in platelets

<p>Abstract</p> <p>Background</p> <p>The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1^-/-mice.</p> <p>Results</p> <p>The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1^-/-mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay.</p> <p>Conclusion</p> <p>This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.</p

Pre-M Phase-promoting Factor Associates with Annulate Lamellae in Xenopus Oocytes and Egg Extracts

We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates

HS1, a Lyn Kinase Substrate, Is Abnormally Expressed in B-Chronic Lymphocytic Leukemia and Correlates with Response to Fludarabine-Based Regimen

In B-Chronic Lymphocytic Leukemia (B-CLL) kinase Lyn is overexpressed, active, abnormally distributed, and part of a cytosolic complex involving hematopoietic lineage cell-specific protein 1 (HS1). These aberrant properties of Lyn could partially explain leukemic cells’ defective apoptosis, directly or through its substrates, for example, HS1 that has been associated to apoptosis in different cell types. To verify the hypothesis of HS1 involvement in Lyn-mediated leukemic cell survival, we investigated HS1 protein in 71 untreated B-CLL patients and 26 healthy controls. We found HS1 overexpressed in leukemic as compared to normal B lymphocytes (1.38±0.54 vs 0.86±0.29, p<0.01), and when HS1 levels were correlated to clinical parameters we found a higher expression of HS1 in poor-prognosis patients. Moreover, HS1 levels significantly decreased in ex vivo leukemic cells of patients responding to a fludarabine-containing regimen. We also observed that HS1 is partially localized in the nucleus of neoplastic B cells. All these data add new information on HS1 study, hypothesizing a pivotal role of HS1 in Lyn-mediated modulation of leukemic cells’ survival and focusing, one more time, the attention on the BCR-Lyn axis as a putative target for new therapeutic strategies in this disorder

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster

Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

SNARE complex assembly and mobilization of GLUT4 vesicles is coordinated through direct targeting of Munc18c by the insulin receptor tyrosine kinase