Use of polyamidoamine dendrimers to engineer BDNF-producing human mesenchymal stem cells

We report the use of polyamidoamine (PAMAM-NH₂) dendrimers along with other non-viral vehicles for the in vitro transfection of human bone marrow mesenchymal stem cells (hMSCs) and for engineering MSCs to secrete brain-derived neurotrophic factor (BDNF). Different generations of cationic polyamidoamine dendrimers (generations 3–6) were tested on HEK 293T cells. hMSCs were then transfected with PAMAM-NH₂ G4 dendrimers and Lipofectamine 2000, which elicited the expression of GFP reporter in around 6 and 20% of the cells, respectively. Both vehicles were then shown to elicit the expression of BDNF in MSCs from a bicistronic cassette. Non-virally induced neurotrophin expression may be a safe and easy method for adapting autologous stem cells for therapeutic treatment of diseases and neural system injuries.     

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC₅₀ values below 1 μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC₅₀ values lying in a range of 11–45 nM. These most potent compounds might be bi-substrate analogues.     

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Univalent binding of the Cry1Ab toxin of Bacillus thuringiensis to a conserved structural motif in the cadherin receptor BT-R1

The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R1, a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S., Midboe, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis, Insect Biochem. Mol. Biol. 32, 1025-1036]. BT-R1 represents a family of invertebrate cadherins whose ectodomains (ECs) are composed of multiple cadherin repeats (EC1 through EC12). In the present work, we determined the Cry1Ab toxin binding site in BT-R1 in the context of cadherin structural determinants. Our studies revealed a conserved structural motif for toxin binding that includes two distinct regions within the N- and C-termini of EC12. These regions are characterized by unique sequence signatures that mark the toxin-binding function in BT-R1 as well as in homologous lepidopteran cadherins. Structure modeling of EC12 discloses the conserved motif as a single broad interface that holds the N- and C-termini in close proximity. Binding of toxin to BT-R1, which is univalent, and the subsequent downstream molecular events responsible for cell death depend on the conserved motif in EC12.     

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.     

Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.     

Pre-steady-state Kinetic and Structural Analysis of Interaction of Methionine γ-Lyase from Citrobacter freundii with Inhibitors

Methionine γ-lyase (MGL) catalyzes the γ-elimination of l-methionine and its derivatives as well as the β-elimination of l-cysteine and its analogs. These reactions yield α-keto acids and thiols. The mechanism of chemical conversion of amino acids includes numerous reaction intermediates. The detailed analysis of MGL interaction with glycine, l-alanine, l-norvaline, and l-cycloserine was performed by pre-steady-state stopped-flow kinetics. The structure of side chains of the amino acids is important both for their binding with enzyme and for the stability of the external aldimine and ketimine intermediates. X-ray structure of the MGL·l-cycloserine complex has been solved at 1.6 Å resolution. The structure models the ketimine intermediate of physiological reaction. The results elucidate the mechanisms of the intermediate interconversion at the stages of external aldimine and ketimine formation.     

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 ? resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.     

Productive replication of human adenovirus type 5 in canine cells

Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.