Production of Recombinant Human Factor VIII in Different Cell Lines and the Effect of Human XBP1 Co-Expression

Recombinant factor VIII is one of the most complex mammalian proteins and a biotechnology venture required for the treatment of hemophilia A. The complexity of the protein, post-translational modifications and limitations of expression elements make the production of active recombinant FVIII a challenge. Here we report the production of biologically active Factor VIII in two different cell lines, CHO and HepG2, by transient transfection. Two expression vectors based on the CMV promoter were used: one harboring CMV Intron A (InA) and the other without it. To bypass difficulties in secretion, we also studied the influence of co-expression of the human splice isoform of the XBP1 gene. We report the production of recombinant FVIII possessing bioengineered FVIII heavy and light chains, linked by a minimal B domain. In our study, HepG2, a human hepatocyte cell line, expressed Factor VIII ten-fold more than a CHO cell line, and in HepG2 cells, the expression of XBP1 improved Factor VIII activity. For CHO cells, expression was improved by the presence of InA, but no further improvement was noted with XBP1 co-expression. These data suggest that the minimal B domain rFVIII preserves Factor VIII biological activity and that different expression elements can be used to improve its production.     

Binding citrate/DNA in presence of divalent cations – Potential mimicry of acidic peptides/DNA interactions

Citric acid whose structure is comparable to that of small acidic peptides, can bind to DNA in the presence of divalent cations (Cu²⁺, Fe²⁺, Zn²⁺, Mg²⁺). Citrate-DNA interaction occurs also in a cell homogenate and in this experimental model too requires the presence of natural divalent cations. In fact the addition of 2 mM EDTA to cell homogenate strongly decreases the DNA-citrate binding. The results demonstrate that divalent cations can act as bridges between two acidic molecules and that citric acid can mimic the structure of acidic peptides.     

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients

Background  

The routine determination of drug resistance in newly HIV-1 infected individuals documents a potential increase in the transmission of drug-resistant variants. Plasma samples from twenty seven therapy naive HIV-1 infected Italian patients were analyzed by the line probe assay (LIPA) and the TruGene HIV-1 assay for the detection of mutations conferring resistance to HIV-1.     

High-performance liquid chromatographic determination of diclofenac in human plasma after solid-phase extraction

A novel high-performance liquid chromatographic (HPLC) method for the quantification of diclofenac in human plasma was set up. Samples, added with ibuprofen (used as internal standard) were purified by solid-phase extraction using Abselut Nexus cartridges (Varian) not requiring pre-conditioning. Drugs of interest were eluted directly into the autosampler vials and injected. The recovery of diclofenac was 92%, the analysis lasted 7 min with a sensitivity of 5 ng/ml and intra- and inter-day RSDs of 3 and 8%, respectively. The pharmacokinetics of diclofenac after oral and rectal administration in 10 healthy volunteers are reported.     

Lipoteichoic acid-induced lung inflammation depends on TLR2 and the concerted action of TLR4 and the platelet-activating factor receptor

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.     

A single mutation in the IF3 N-terminal domain perturbs the fidelity of translation initiation at three levels

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3_Y75N protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed.     

Response of calcareous nannofossils to the Paleocene–Eocene Thermal Maximum: Observations on composition,preservation and calcification in sediments from ODP Site 1263 (Walvis Ridge — SW Atlantic)

In this study we present the results of a detailed analysis on calcareous nannofossil assemblages from sediment cores of ODP Site 1263 (Southern East Atlantic, Walvis Ridge). This section represents one of the few complete deep-sea sections that document the Paleocene–Eocene Thermal Maximum (PETM) in the pelagic realm. The PETM transient event was characterized by a brief, but intense interval of global warming, a global negative carbon isotope excursion (CIE), and widespread dissolution of seafloor carbonate sediments. Paired analysis at polarizing light microscope (LM) and Scanning Electron Microscope (SEM) documents the different “behavior” of nannofossils through the different phases of the PETM, at the onset of CIE, within the CIE, and during the recovery interval. The presence of anomalous specimens and morphotypes within some nannofossil taxa, recorded during previous LM high resolution analyses, has been further investigated in selected samples at the SEM. Besides the known representatives of the CIE-PETM “excursion nanno-flora”, as Rhomboaster calcitrapa group and Discoaster anartios, the analysis revealed the presence of peculiar morphotypes of Fasciculithus and deformed specimens of Discoaster nobilis group, Discoaster mediosus and Discoaster multiradiatus that are considered related to the anomalous amount of CO₂ in the ocean-atmosphere system during the early phase of PETM. Comparative analyses were performed in few selected samples from other PETM sections located at different latitudes in the Atlantic and Pacific oceans. Although the anomalous geochemical conditions during the PETM-CIE interval seem to have had some influence on the nannofossil production, calcification and assemblage composition, it results that local productivity together with post depositional (diagenetic) conditions were additional important controlling factors on nannofossil assemblages. Preliminary data from Eocene Thermal Maximum 2 (ETM2 or Elmo) suggest that nannofossil malformations are not exclusive of the PETM, and are associated to other episodes of perturbation of the C cycle.