Confirmation of operative acetate, H2/CO2 and methanol methanogenic pathways in the solid-state refuse fermentation

By use of the radiolabelled substrates sodium [1–¹⁴C] acetate, sodium [2–¹⁴C] acetate, NaH¹⁴CO₃ and ¹⁴CH₃OH, three of the possible methanogenic pathways in fermenting refuse were confirmed. Due to the absence of a methanol pool, however, the relative contribution of each could not be determined. Circumstantial evidence for an operative trimethylamine pathway was gained but not confirmed whilst preliminary attempts to stimulate methanogenesis in refuse by supplementation with mono-and dimethylamine proved unsuccessful.     

Urinary proteins with pro-apoptotic and antitumor activity

Analysis of the protein composition of urine has been the subject of much research that has captured the interest of scientific groups over the years. A number of factors have been isolated from urine that possess anti-neoplastic activities as seen both in vitro and in vivo studies. The urine from pregnant women and commercial preparations of crude clinical grade human chorionic gonadotropin contain factors (HAF for hCG associated factor) with anti-Kaposi's sarcoma activity. Also found in urine with activity are eosinophil-derived neurotoxin (EDN), anti-neoplastic urinary protein (ANUP), inhibin, activin A, and angiostatin. The anti-cancer activity of urinary proteins is associated with apoptosis of endothelial cells and of tumor-associated endothelial cells. A better understanding of the biological functions of these various urinary proteins, and of others that remain to be discovered, should provide insights into novel cell regulatory systems operating during pregnancy.     

The D246V mutant of DNA polymerase beta misincorporates nucleotides: evidence for a role for the flexible loop in DNA positioning within the active site

DNA polymerase beta, a member of the X family of DNA polymerases, is known to be involved in base excision repair. A key to determining the biochemical properties of this DNA polymerase is structure-function studies of site-specific mutants that result in substitution of particular amino acids at critical sites. In a previous genetic screen, we identified three 3'-azido-2',3'-dideoxythymidine 5'-triphosphate-resistant mutants, namely E249K, D246V, and R253M, of polymerase beta in the flexible loop of the palm domain. In this work, we perform an extensive kinetic analysis to investigate the role of the D246V mutant on polymerase fidelity. We find that D246V misincorporates T opposite template bases G and C. The mechanistic basis of misincorporation appears to be altered DNA positioning within the active site. We provide evidence that the fidelity of D246V is greatly affected by the base that is 5' of the templating base. We propose that the Asp residue at position 246 helps to maintain the proper positioning of the DNA within the polymerase active site and maintains the fidelity of polymerase beta. Altogether, the results suggest that the flexible loop domain of polymerase beta plays a major role in its fidelity.     

Target selectivity in EF-hand calcium binding proteins

EF-hand calcium binding proteins have remarkable sequence homology and structural similarity, yet their response to binding of calcium is diverse and they function in a wide range of biological processes. Knowledge of the fine-tuning of EF-hand protein sequences to optimize specific biochemical properties has been significantly advanced over the past 10 years by determination of atomic resolution structures. These data lay the foundation for addressing how functional selectivity is generated from a generic ionic signal. This review presents current ideas about the structural mechanisms that provide the selectivity of different EF-hand proteins for specific cellular targets, using S100 and calmodulin family proteins to demonstrate the critical concepts. Three factors contribute significantly to target selectivity: molecular architecture, response to binding of Ca(2+) ions, and the characteristics of target binding surfaces. Comparisons of calmodulin and S100 proteins provide insights into the role these factors play in facilitating the variety of binding configurations necessary for recognizing a diverse set of targets.     

Tumorigenesis by human herpesvirus 8 vGPCR is accelerated by human immunodeficiency virus type 1 Tat

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-kappaB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1 tat (but not a transactivation-defective mutant) into these tumor cells increases NF-kappaB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.     

Novel structures of self-associating stapled peptides

Hydrocarbon stapling of peptides is a powerful technique to transform linear peptides into cell-permeable helical structures that can bind to specific biological targets. In this study, we have used high resolution solution NMR techniques complemented by dynamic light scattering to characterize extensively a family of hydrocarbon stapled peptides with known inhibitory activity against HIV-1 capsid assembly to evaluate the various factors that modulate activity. The helical peptides share a common binding motif but differ in charge, the length, and position of the staple. An important outcome of the study was to show the peptides, share a propensity to self-associate into organized polymeric structures mediated predominantly by hydrophobic interactions between the olefinic chain and the aromatic side-chains from the peptide. We have also investigated in detail the structural significance of the length and position of the staple, and of olefinic bond isomerization in stabilizing the helical conformation of the peptides as potential factors driving polymerization. This study presents the numerous challenges of designing biologically active stapled peptides and the conclusions have broad implications for optimizing a promising new class of compounds in drug discovery.     

Implications on zinc binding to S100A2

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.     

FANCD2 influences replication fork processes and genome stability in response to clustered DSBs

Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.