Anti-idiotype x anti-LFA-1 bispecific antibodies inhibit metastasis of B cell lymphoma

Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id x anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.     

Grape berry position affects the diurnal dynamics of its metabolic profile

Solar irradiance and air temperature are characterized by dramatic circadian fluctuations and are known to significantly modulate fruit composition. To date, it remains unclear whether the abrupt, yet predictive, diurnal changes in radiation and temperature prompt direct metabolic turn‐over in the fruit. We assessed the role of fruit insolation, air temperature, and source‐tissue CO₂ assimilation in the diurnal compositional changes in ripening grape berries. This was performed by comparing the diurnal changes in metabolite profiles of berries positioned such that they experienced (a) contrasting diurnal solar irradiance patterns, and (b) similar irradiance but contrasting diurnal CO₂ assimilation patterns of adjacent leaves. Grape carbon levels increased during the morning and decreased thereafter. Sucrose levels decreased throughout the day and were correlated with air temperature, but not with the diurnal pattern of leaf CO₂ assimilation. Tight correlation between sucrose and glucose‐6‐phosphate indicated the involvement of photorespiration/glycolysis in sucrose depletion. Amino acids, polyamines, and phenylpropanoids fluctuated diurnally, and were highly responsive to the diurnal insolation pattern of the fruit. Our results fill the knowledge gap regarding the circadian pattern of source‐sink assimilate‐translocation in grapevine. In addition, they suggest that short‐term direct solar exposure of the fruit impacts both its diurnal and nocturnal metabolism.     

The role of cytosolic phospholipase A2α in amyloid precursor protein induction by amyloid beta1‐42: implication for neurodegeneration

Amyloid‐β peptides generated by proteolysis of the β‐amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A₂α (cPLA₂α) plays a role in elevated APP protein expression induced by aggregated amyloid‐β_1‐42 (Aβ) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA₂α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aβ for 24 h and in cortical neuron of human amyloid‐β_1‐42 brain infused mice. Prevention of cPLA₂α up‐regulation and its activity by oligonucleotide antisense against cPLA₂α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA₂α in the signals leading to APP induction, increased cPLA₂α expression and activity induced by Aβ was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase‐2 and the production of prostaglandin E₂ (PGE₂) were prevented. Addition of PGE₂ or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H‐89) attenuated the elevation of APP induced by Aβ. Inhibition of elevated cPLA₂α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aβ which coincided with cPLA₂α dependent activation of CREB in the cortex of Aβ brain infused mice. Our results show that accumulation of Aβ induced elevation of APP protein expression mediated by cPLA₂α, PGE₂ release, and CREB activation via protein kinase A pathway.

     

Targeting Enterococcus faecalis Biofilms with Phage Therapy

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KP339049","term_id":"761545084","term_text":"KP339049"}}KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.     

Aberrant Apoptotic Response of Colorectal Cancer Cells to Novel Nucleoside Analogues

Despite the increased understanding of colorectal cancer and the introduction of targeted drug therapy, the metastatic phase of the disease remains refractory to treatment. Since the deregulation of normal apoptosis contributes to the pathogenesis of colorectal cancer, novel nucleoside analogues were synthesized here and evaluated for their ability to induce apoptosis and cause cell death in two colorectal adeno-carcinoma cell lines, Caco-2 and HT-29. Three novel nucleoside analogues assessed here showed cytotoxic activity, as measured by the MTT assay against both cell lines: the IC₅₀ values ranged between 3 and 37 μM, with Caco-2 cells being more sensitive than HT-29 cells. Compared to camptothecin, the positive control, the nucleoside analogues were significantly less toxic to normal unstimulated leukocytes (p>0.05). Moreover, the nucleosides were able to induce apoptosis as measured by an increase in caspase 8 and caspase 3 activity above that of the control. This was additionally supported by data derived from Annexin V-FITC assays. Despite marginal changes to the mitochondrial membrane potential, all three nucleosides caused a significant increase in cytosolic cytochrome c (p>0.05), with a corresponding decrease in mitochondrial cytochrome c. Morphological analysis of both cell lines showed the rapid appearance of vacuoles following exposure to two of the nucleosides, while a third caused cellular detachment, delayed cytoplasmic vacuolisation and nuclear abnormalities. Preliminary investigations, using the autophagic indicator monodansylcadaverine and chloroquine as positive control, showed that two of the nucleosides induced the formation of autophagic vacuoles. In summary, the novel nucleoside analogues showed selective cytotoxicity towards both cancer cell lines and are effective initiators of an unusual apoptotic response, demonstrating their potential to serve as structural scaffolds for more potent analogues.     

Principles of nanostructure design with protein building blocks

Currently there is increasing interest in nanostructures and their design. Nanostructure design involves the ability to predictably manipulate the properties of the self-assembly of autonomous units. Autonomous units have preferred conformational states. The units can be synthetic material science-based or derived from functional biological macromolecules. Autonomous biological building blocks with available structures provide an extremely rich and useful resource for design. For proteins, the structural databases contain large libraries of protein molecules and their building blocks with a range of shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these can expand the available chemical space and enhance the desired properties. Here we focus on the principles of nanostructure design with protein building blocks.     

Stability of tubular structures based on beta-helical proteins: self-assembled versus polymerized nanoconstructs and wild-type versus mutated sequences

In this work we used atomistic molecular dynamics simulations to examine different aspects of tubular nanostructures constructed using protein building blocks with a beta-helical conformation. Initially, we considered two different natural protein building blocks, which were extracted from the protein data base, to compare the relative stabilities of the nanotubes obtained made of self-assembled and covalently linked repeats. Results show nanotubes constructed by linking building blocks through covalent bonds are very stable suggesting that the basic principles of polymer physics are valid when the repeating units are made of large fragments of proteins. In contrast, the stability of self-assembled nanostructures strongly depends on the attractive nonbonding interactions associated to building blocks aligned in a complementary manner. On the other hand, we investigated the ability of a conformationally constrained synthetic amino acid to enhance the stability of both self-assembled and polymerized nanotubes when it is used to substitute natural residues. Specifically, we considered 1-aminocyclopentane-1-caboxylic acid, which involves strong stereochemical constraints produced by the cyclopentane side chain. We found that the incorporation of this amino acid within the more flexible regions of the beta-helical building blocks is an excellent strategy to enhance the stability of the nanotubes. Thus, when a single mutation is performed in the loop region of the beta-helix, the bend architecture of the whole loop is stabilized since the conformational mobility is reduced not only at the mutated position but also at the adjacent positions.     

A novel substrate mimetic inhibitor of PKB/Akt inhibits prostate cancer tumor growth in mice by blocking the PKB pathway

We describe a novel, potent peptide substrate mimetic inhibitor of protein kinase B (PKB/Akt). The compound selectively kills prostate cancer cells, in which PKB is highly activated, but not normal cells, or cancer cells in which PKB is not activated. The inhibitor induces apoptosis and inhibits the phosphorylation of PKB substrates in prostate cancer cell lines and significantly increases the efficacy of chemotherapy agents to induce prostate cancer cell death, when given in combination. In vivo, the inhibitor exhibits a strong antitumor effect in two prostate cancer mouse models. Moreover, treated animals develop significantly less lung metastases compared to untreated ones, and the effect is accompanied by a significant decrease in blood PSA [prostate-specific antigen] levels in treated animals. This compound and its potential analogues may be developed into novel, potent, and safe anticancer agents, both as stand-alone treatment and in combination with other chemotherapy agents.