Conversion of the Kunitz-type module of collagen VI into a highly active trypsin inhibitor by site-directed mutagenesis.

The recombinant Kunitz protease inhibitor module (domain C5) of human collagen α3(VI) chain was previously shown to lack inhibitory activity for proteases with trypsin-like specificity and some other proteases. We have now prepared mutants in the binding loop region including the P1′ site (D2889 → A), the P2′ site (F2890 → R) and the P3 site (T2886 → P) and in a more remote region (W2907 → V) either as individual substitutions or combinations of them. These mutants were analyzed for their kinetics of binding to trypsin by surface plasmon resonance and for their capacity to inhibit various proteases. Single substitutions (D → A, T → P, W → V) showed an effect only for D → A which bound to trypsin with Kd= 0.25 μM. A 25–100-fold increase in affinity was observed for the double mutants T → P/D → A and F → R/D → A and approached the affinity of aprotinin (Kd≈0.01 nM) in two different triple mutants. These affinities correlated well with the inhibitory capacities of the mutants for trypsin in the cleavage of a large protein and a small peptide substrate. A similar but not completely identical improvement in inhibitory capacity was also observed for leucocyte elastase but not for thrombin. These data could be interpreted in terms of steric interferences or lack of hydrogen bonding of a few critical residues based on three-dimensional structures available for the C5 domain

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

Validation of the revised IPSS at transplant in patients with myelodysplastic syndrome/transformed acute myelogenous leukemia receiving allogeneic stem cell transplantation

The International Prognostic Scoring System has been revised (IPSS-R) to predict prognosis of patients with myelodysplastic syndromes at diagnosis. To validate the use of the IPSS-R assesse

Cytoplasmic p21(WAF1/CIP1 )expression is correlated with HER-2/ neu in breast cancer and is an independent predictor of prognosis

BACKGROUND: HER-2 (c-erbB2/Neu) predicts the prognosis of and may influence treatment responses in breast cancer. HER-2 activity induces the cytoplasmic location of p21(WAFI/CIPI )in cell culture, accompanied by resistance to apoptosis. p21(WAFI/CIPI )is a cyclin-dependent kinase inhibitor activated by p53 to produce cell cycle arrest in association with nuclear localisation of p21(WAFI/CIPI). We previously showed that higher levels of cytoplasmic p21(WAFI/CIPI )in breast cancers predicted reduced survival at 5 years. The present study examined HER-2 and p21(WAFI/CIPI )expression in a series of breast cancers with up to 9 years of follow-up, to evaluate whether in vitro findings were related to clinical data and the effect on outcome. METHODS: The CB11 anti-HER2 monoclonal antibody and the DAKO Envision Plus system were used to evaluate HER-2 expression in 73 patients. p21(WAFI/CIPI )staining was performed as described previously using the mouse monoclonal antibody Ab-1 (Calbiochem, Cambridge, MA, USA). RESULTS: HER-2 was evaluable in 67 patients and was expressed in 19% of cases, predicting reduced overall survival (P = 0.02) and reduced relapse-free survival (P = 0.004; Cox regression model). HER-2-positive tumours showed proportionately higher cytoplasmic p21(WAFI/CIPI )staining using an intensity distribution score (median, 95) compared with HER-2-negative cancers (median, 47) (P = 0.005). There was a much weaker association between nuclear p21(WAFI/CIPI )and HER-2 expression (P = 0.05), suggesting an inverse relationship between nuclear p21(WAF1/CIP1 )and HER-2. CONCLUSION: This study highlights a new pathway by which HER-2 may modify cancer behaviour. HER-2 as a predictor of poor prognosis may partly relate to its ability to influence the relocalisation of p21(WAFI/CIPI )from the nucleus to the cytoplasm, resulting in a loss of p21(WAFI/CIPI)tumour suppressor functions. Cytoplasmic p21(WAFI/CIPI )may be a surrogate marker of functional HER-2 in vivo

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent

[Background] Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO).[Methods] Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining.Peer reviewe

The MLL recombinome of acute leukemias in 2017

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients

Alternative splicing of barley clock genes in response to low temperature:evidence for alternative splicing conservation

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement

Epidemiological, genetic, and clinical characterization by age of newly diagnosed acute myeloid leukemia based on an academic population-based registry study (AMLSG BiO)

We describe genetic and clinical characteristics of acute myeloid leukemia (AML) patients according to age from an academic population-based registry. Adult patients with newly diagnosed AML at 63 centers in Germany and Austria were followed within the AMLSG BiO registry (NCT01252485). Between January 1, 2012, and December 31, 2014, data of 3525 patients with AML (45% women) were collected. The median age was 65 years (range 18–94). The comparison of age-specific AML incidence rates with epidemiological cancer registries revealed excellent coverage in patients 0 were associated with non-intensive treatment or best supportive care. The AMLSG BiO registry provides reliable population-based distributions of genetic, clinical, and treatment characteristics according to age

Simultaneous observations of PKS 2155-304 with H.E.S.S., Fermi, RXTE and ATOM: spectral energy distributions and variability in a low state

We report on the first simultaneous observations that cover the optical, X-ray, and high energy gamma-ray bands of the BL Lac object PKS 2155-304. The gamma-ray bands were observed for 11 days, between 25 August and 6 September 2008, jointly with the Fermi Gamma-ray Space Telescope and the H.E.S.S. atmospheric Cherenkov array, providing the first simultaneous MeV-TeV spectral energy distribution with the new generation of gamma-ray telescopes. The ATOM telescope and the RXTE and Swift observatories provided optical and X-ray coverage of the low-energy component over the same time period. The object was close to the lowest archival X-ray and Very High Energy state, whereas the optical flux was much higher. The light curves show relatively little (~30%$) variability overall when compared to past flaring episodes, but we find a clear optical/VHE correlation and evidence for a correlation of the X-rays with the high energy spectral index. Contrary to previous observations in the flaring state, we do not find any correlation between the X-ray and VHE components. Although synchrotron self-Compton models are often invoked to explain the SEDs of BL Lac objects, the most common versions of these models are at odds with the correlated variability we find in the various bands for PKS 2155-304.Comment: Accepted for publication in the Astrophysical Journa