Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

High-precision tumor targeting with conventional therapeutics is based on the concept of the ideal drug as a "magic bullet"; this became possible after techniques were developed for production of monoclonal antibodies (mAbs). Innovative DNA technologies have revolutionized this area and enhanced clinical efficiency of mAbs. The experience of applying small-size recombinant antibodies (monovalent binding fragments and their derivatives) to cancer targeting showed that even high-affinity monovalent interactions provide fast blood clearance but only modest retention time on the target antigen. Conversion of recombinant antibodies into multivalent format increases their functional affinity, decreases dissociation rates for cell-surface and optimizes biodistribution. In addition, it allows the creation of bispecific antibody molecules that can target two different antigens simultaneously and do not exist in nature. Different multimerization strategies used now in antibody engineering make it possible to optimize biodistribution and tumor targeting of recombinant antibody constructs for cancer diagnostics and therapy.     

Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.     

Phototoxic effects of fluorescent protein KillerRed on tumor cells in mice

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phloretin-Induced Reduction in Dipole Potential of Sterol-Containing Bilayers

The phloretin-induced reduction in the dipole potential of planar lipid bilayers containing cholesterol, ergosterol, stigmasterol, 7-dehydrocholesterol and 5α-androstan-3β-ol was investigated. It is shown that effects depend on the type and concentration of membrane sterol. It is supposed that the effectiveness of phloretin in reducing the dipole potential of the bilayers that contain cholesterol, ergosterol and 7-dehydrocholesterol correlates with the ordering and condensing effects. The role of the concentration-dependent ability of different sterols to promote lateral heterogeneity in membranes is also discussed.     

Use of Vocal Fingerprinting for Specific Discrimination of Gray (Microcebus murinus) and Rufous Mouse Lemurs (Microcebus rufus)

Advertisement calls are often important noninvasive tools for discriminating cryptic species and for assessing specific diversity and speciation patterns in nature. We investigated the contribution of these calls to uncover specific diversity in nocturnal Malagasy lemurs. We compared sexual advertisement and predator advertisement calls of two mouse lemur species, western gray and eastern rufous mouse lemurs (Microcebus murinus and M. rufus, respectively) living in two contrasting habitats (dry deciduous vs. rain forest), and analyzed them statistically. Both species emitted several highly variable whistle calls in the context of predator-avoidance. Intrapopulation variation was high and overlapped interspecific variation. Sexual advertisement calls, given in the mating context, displayed a totally distinct, species-specific acoustic structure. Whereas gray mouse lemurs produced rapidly multifrequency modulated, long trill calls, rufous mouse lemurs gave slowly frequency-modulated short chirp calls. Our results suggest specific status for gray and rufous mouse lemurs and indicate the importance of predation and social needs in shaping vocal communication.     

Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1

Herpesviruses enter cells by membrane fusion either at the plasma membrane or in endosomes, depending on the cell type. Glycoprotein B (gB) is a conserved component of the multiprotein herpesvirus fusion machinery and functions as a fusion protein, with two internal fusion loops, FL1 and FL2. We determined the crystal structures of the ectodomains of two FL1 mutants of herpes simplex virus type 1 (HSV-1) gB to clarify whether their fusion-null phenotypes were due to global or local effects of the mutations on the structure of the gB ectodomain. Each mutant has a single point mutation of a hydrophobic residue in FL1 that eliminates the hydrophobic side chain. We found that neither mutation affected the conformation of FL1, although one mutation slightly altered the conformation of FL2, and we conclude that the fusion-null phenotype is due to the absence of a hydrophobic side chain at the mutated position. Because the ectodomains of the wild-type and the mutant forms of gB crystallized at both low and neutral pH, we were able to determine the effect of pH on gB conformation at the atomic level. For viruses that enter cells by endocytosis, the low pH of the endosome effects major conformational changes in their fusion proteins, thereby promoting fusion of the viral envelope with the endosomal membrane. We show here that upon exposure of gB to low pH, FL2 undergoes a major relocation, probably driven by protonation of a key histidine residue. Relocation of FL2, as well as additional small conformational changes in the gB ectodomain, helps explain previously noted changes in its antigenic and biochemical properties. However, no global pH-dependent changes in gB structure were detected in either the wild-type or the mutant forms of gB. Thus, low pH causes local conformational changes in gB that are very different from the large-scale fusogenic conformational changes in other viral fusion proteins. We propose that these conformational changes, albeit modest, play an important functional role during endocytic entry of HSV.     

PERK utilizes intrinsic lipid kinase activity to generate phosphatidic acid, mediate Akt activation, and promote adipocyte differentiation

The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

The effect of electrodialytic treatment and Na2H2EDTA addition on methanogenic activity of copper-amended anaerobic granular sludge: treatment costs and energy consumption

The effect of electrodialytic treatment in terms of a current density, pH and Na₂H₂EDTA addition on the methanogenic activity of copper-amended anaerobic granular sludge taken from the UASB reactor from paper mill was evaluated. Moreover, the specific energy consumption and simplified operational and treatment costs were calculated. Addition of Na₂H₂EDTA (at pH 7.7) to copper-amended sludge resulted in the highest microbial activity (62 mg CH₄-COD g VSS⁻¹ day⁻¹) suggesting that Na₂H₂EDTA decreased the toxic effects of copper on the methanogenic activity of the anaerobic granular sludge. The highest methane production (159 %) was also observed upon Na₂H₂EDTA addition and simultaneous electricity application (pH 7.7). The energy consumption during the treatment was 560, 840, 1400 and 1680 kW h m⁻³ at current densities of 0.23, 0.34, 0.57 and 0.69 mA cm⁻², respectively. This corresponded to a treatment costs in terms of electricity expenditure from 39.2 to 117.6 € per cubic meter of sludge.