A human ubiquitin conjugating enzyme,L-UBC,maps in the Alzheimer's disease locus on Chromosome 14q24.3

We have identified a novel ubiquitin conjugating enzyme gene, L-UBC, which maps to human Chromosome (Chr) 14q24.3. This is also the location of the major early onset familial Alzheimer's disease gene (FAD3). L-UBC encodes a protein that demonstrates homology to the yeast ubiquitin conjugating enzyme, UBC-4, and human UbcH5. Their functions are to ubiquitinate specific proteins targeted for degradation. The protein also exhibits very strong homology to a rabbit protein, E2-F1, which mediates p53 degradation driven by papilloma virus E6 protein in vitro. The accumulation of specific proteins that have undergone aberrant processing in neurofibrillary tangles and amyloid plaques is the classic pathological feature in brains of Alzheimer's disease patients. Abnormal ubiquitination has previously been suggested to play a role in the etiology of Alzheimer's disease. This gene therefore represents a plausible candidate gene for FAD3.     

ALPPS Procedure for Extended Liver Resections: A Single Centre Experience and a Systematic Review

Aim

To report a single-centre experience with the novel Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) technique and systematically review the related literature.

Methods

Since January 2013, patients with extended primary or secondary liver tumors whose future liver remnant (FLR) was considered too small to allow hepatic resection were prospectively assessed for the ALPPS procedure. A systematic literature search was performed using PubMed, Scopus and the Cochrane Library Central.

Results

Until July 2014 ALPPS was completed in 9 patients whose mean age was 60±8 years. Indications for surgical resection were metastases from colorectal cancer in 3 cases, perihilar cholangiocarcinoma in 3 cases, intrahepatic cholangiocarcinoma in 2 cases and hepatocellular carcinoma without chronic liver disease in 1 case. The calculated FLR volume was 289±122 mL (21.1±5.5%) before ALPPS-1 and 528±121 mL (32.2±5.7%) before ALLPS-2 (p<0.001). The increase in FLR between the two procedures was 96±47% (range: 24–160%, p<0.001). Additional interventions were performed in 4 cases: 3 patients underwent Roux-en-Y hepaticojejunostomy, and one case underwent wedge resection of a residual tumor in the FLR. The average time between the first and second step of the procedure was 10.8±2.9 days. The average hospital stay was 24.1±13.3 days. There was 1 postoperative death due to hepatic failure in the oldest patient of this series who had a perihilar cholangiocarcinoma and concomitant liver fibrosis; 11 complications occurred in 6 patients, 4 of whom had grade III or above disease. After a mean follow-up of 17.1±8.5 months, the overall survival was 89% at 3–6 and 12 months. The recurrence-free survival was 100%, 87.5% and 75% at 3-6-12 months respectively. The literature search yielded 148 articles, of which 22 articles published between 2012 and 2015 were included in this systematic review.

Conclusion

The ALPPS technique effectively increased the resectability of otherwise inoperable liver tumors. The postoperative morbidity in our series was high in accordance with the data from the systematic review. Age, liver fibrosis and presence of biliary stenting were predisposing factors for postoperative morbidity and mortality.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Role of the protein in the DNA sequence specificity of the cleavage site stabilized by the camptothecin topoisomerase IB inhibitor: a metadynamics study

Camptothecin (CPT) is a topoisomerase IB (TopIB) selective inhibitor whose derivatives are currently used in cancer therapy. TopIB cleaves DNA at any sequence, but in the presence of CPT the only stabilized protein–DNA covalent complex is the one having a thymine in position −1 with respect to the cleavage site. A metadynamics simulation of two TopIB–DNA–CPT ternary complexes differing for the presence of a thymine or a cytosine in position −1 indicates the occurrence of two different drug’s unbinding pathways. The free-energy difference between the bound state and the transition state is large when a thymine is present in position −1 and is strongly reduced in presence of a cytosine, in line with the different drug stabilization properties of the two systems. Such a difference is strictly related to the changes in the hydrogen bond network between the protein, the DNA and the drug in the two systems, indicating a direct role of the protein in determining the specificity of the cleavage site sequence stabilized by the CPT. Calculations carried out in presence of one compound of the indenoisoquinoline family (NSC314622) indicate a comparable energy difference between the bound and the transition state independently of the presence of a thymine or a cytosine in position −1, in line with the experimental results.     

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.     

Resistive exercises, with or without whole body vibration, prevent vertebral marrow fat accumulation during 60 days of head-down tilt bed rest in men

Fat accumulates in the bone marrow of lumbar vertebrae with bed rest. Exercise with or without whole body vibration may counter this effect. Our objectives were to measure 1) the vertebral fat fraction (VFF) of men subjected to bed rest who performed resistive exercises with (RVE, n = 7) or without whole body vibration(RE, n = 8) or no exercise (CTR, n = 9) using three MRI techniques; and 2) changes in peripheral blood counts. Twenty-four healthy men (age: 20-45 yr) underwent -6° head-down tilt (HDT) bed rest for 60 days. MRI was performed using three techniques (fat saturation, proton spectroscopy, and in and out of phase) to measure the fat fraction of L(3), L(4), and/or L(5) at baseline, mid-HDT, and end-HDT. Erythrocytes and leukocytes were counted at HDT days 19, 33, 47, 54, and 60. The mean absolute VFF was increased in the CTR group at mid-HDT and end-HDT (+3.9 ± 1.3 and +3.6 ± 1.2%, respectively, both P < 0.05). The RE group had a smaller VFF change than the CTR group at mid-HDT (-0.9 ± 1.2 vs. +3.9 ± 1.3%, P < 0.05). The RVE group had a smaller VFF change than the CTR group at end-HDT (-2.6 ± 1.9 vs. +3.5 ± 1.2%, P < 0.05). Erythrocyte counts were increased in all groups at HDT day 19 and HDT day 33 and in the RE group at HDT day 54 (all P < 0.05). Bed rest for 60 days at -6° HDT increased lumbar VFF in men beyond natural involution. RVE and RE regimens effectively prevented VFF accumulation. Higher erythrocyte counts were not altered by RVE or RE. Whole body vibration, along with RE administered to people with prolonged immobility, may prevent fat accumulation in their bone marrow.     

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k_off) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k_off value increases from (1.4 ± 0.2) × 10⁻⁴ s⁻¹, in the absence of the drug, to (9.5 ± 1.2) × 10⁻³ s⁻¹, in the presence of 1.0 × 10⁻² M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K₁ = (3.1 ± 0.4) × 10⁻⁷ M, K₂ = (1.7 ± 0.2) × 10⁻⁴ M, and K₃ = (2.2 ± 0.2) × 10⁻³ M) were determined. The K₃ value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10⁻³ M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.     

Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts

AIMS: Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species. CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics.