Enhanced cytotoxicity of a polymer–drug conjugate with triple payload of paclitaxel

The development of targeting approaches to selectively release chemotherapeutic drugs into malignant tissue is a major challenge in anticancer therapy. We have synthesized an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer–drug conjugate with an AB₃ self-immolative dendritic linker. HPMA copolymers are known to accumulate selectively in tumors. The water-soluble polymer–drug conjugate was designed to release a triple payload of the hydrophobic drug paclitaxel as a result of cleavage by the endogenous enzyme cathepsin B. The polymer–drug conjugate exhibited enhanced cytotoxicity on murine prostate adenocarcinoma (TRAMP C2) cells in comparison to a classic monomeric drug–polymer conjugate.     

A Compact Representation of Drawing Movements with Sequences of Parabolic Primitives

Some studies suggest that complex arm movements in humans and monkeys may optimize several objective functions, while others claim that arm movements satisfy geometric constraints and are composed of elementary components. However, the ability to unify different constraints has remained an open question. The criterion for a maximally smooth (minimizing jerk) motion is satisfied for parabolic trajectories having constant equi-affine speed, which thus comply with the geometric constraint known as the two-thirds power law. Here we empirically test the hypothesis that parabolic segments provide a compact representation of spontaneous drawing movements. Monkey scribblings performed during a period of practice were recorded. Practiced hand paths could be approximated well by relatively long parabolic segments. Following practice, the orientations and spatial locations of the fitted parabolic segments could be drawn from only 2–4 clusters, and there was less discrepancy between the fitted parabolic segments and the executed paths. This enabled us to show that well-practiced spontaneous scribbling movements can be represented as sequences (“words”) of a small number of elementary parabolic primitives (“letters”). A movement primitive can be defined as a movement entity that cannot be intentionally stopped before its completion. We found that in a well-trained monkey a movement was usually decelerated after receiving a reward, but it stopped only after the completion of a sequence composed of several parabolic segments. Piece-wise parabolic segments can be generated by applying affine geometric transformations to a single parabolic template. Thus, complex movements might be constructed by applying sequences of suitable geometric transformations to a few templates. Our findings therefore suggest that the motor system aims at achieving more parsimonious internal representations through practice, that parabolas serve as geometric primitives and that non-Euclidean variables are employed in internal movement representations (due to the special role of parabolas in equi-affine geometry).     

Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells

The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA-deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non-deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473-an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS-1 phosphorylation, which was dose-dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS-1 via an S6K1-independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on beta-casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells.     

Bacteriophage infection is targeted to cellular poles

The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.     

Chemotherapeutic bone-targeted bisphosphonate prodrugs with hydrolytic mode of activation

Osseous tissues are considered to be limited as therapeutic target sites due to their biological properties. We have designed and synthesized two kinds of hydrolytically activated chemotherapeutic prodrugs containing bisphosphonate, a bone-targeting moiety. The first can be conjugated to drug molecules with an available hydroxy group; the drug is attached to the bisphosphonate component through an ester-labile linkage. The second is for use with drug molecules with amine functional group. In this case, a self-immolative linker is used to attach the drug to the bisphosphonate component through a carbonate-labile linkage. The concept was demonstrated using the drugs camptothecin, which has a hydroxy functional group, and tryptophan, which is a model molecule for a drug with amine functionality. Both prodrugs showed significant binding capability to hydroxyapatite, the major component of bone, and were hydrolytically activated under physiological conditions.     

Functional analysis of novel multidrug transporters from human pathogens

Proteins of the Smr family are the smallest multidrug transporters, about 110 amino acids long, that extrude various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. One of these proteins, EmrE, is an Escherichia coli protein, which has been cloned based on its ability to confer resistance to ethidium and methyl viologen and which has been extensively characterized. More than 60 genes coding for Smr proteins have been identified in several bacteria based on amino acid sequence similarity to the emrE gene. In this work we have analyzed the sequence similarity among these homologues and identified some distinct signature sequence elements and several fully conserved residues. Five of these homologues, from human pathogens Mycobacterium tuberculosis, Bordetella pertussis, and Pseudomonas aeruginosa and from Escherichia coli, were cloned into an E. coli expression system. The proteins were further characterized and show varying degrees of methyl viologen uptake into proteoliposomes and [(3)H]TPP binding in solubilized membranes. The homologues can also form mixed oligomers with EmrE that exhibit intermediate binding characteristics. A comparative study of various homologous proteins provides a tool for deciphering structure-function relationship and monomer-monomer interaction in multidrug transporters and in membrane proteins in general.     

Evolutionary Dynamics of Large <Emphasis Type="Italic">Numts</Emphasis> in the Human Genome: Rarity of Independent Insertions and Abundance of Post-Insertion Duplications

Abstract We determined the phylogenetic positions of 82 large nuclear pseudogenes of mitochondrial origin (numts) within the human genome. For each numt, two possibilities pertaining to its origin were considered: (1) independent insertion from the mitochondria into the nucleus, or (2) genomic duplication subsequent to the insertion. A significant increase in the rate of numt accumulation is seen after the divergence of Platyrrhini (New World monkeys) from the Catarrhini (Old World monkeys, apes and humans). By using pairwise phylogenetic analyses, we were able to demonstrate that this peak in numt accumulation is mostly the result of duplication of preexisting nuclear numts rather than the result of an increase in mitochondrial-sequence insertion. In fact, only about a third of all the numt repertoire in the human nuclear genome is due to insertions of mitochondrial sequences, the rest originated as duplications of preexisting numts. Hence, we conclude that numt insertion occurs at a much lower rate than previously reported. As expected under the assumption that genomic duplications occur at rates that are uninfluenced by content, older numts were found to be duplicated more times than recently inserted ones.     

Insights into the structure and domain flexibility of full-length pro-matrix metalloproteinase-9/gelatinase B

The multidomain zinc endopeptidase matrix metalloproteinase-9 (MMP-9) is a recognized therapeutic target in autoimmune diseases, vascular pathologies, and cancer. Despite its importance, structural characterization of full-length pro-MMP-9 is incomplete. Here, we report the structural model of full-length pro-MMP-9 and, in particular, the molecular character of its unique proline-rich and heavily O-glycosylated (OG) domain. Using a powerful combination of small-angle X-ray scattering and single-molecule imaging, we demonstrate that pro-MMP-9 possesses an elongated structure with two terminal globular domains connected by an unstructured OG domain. Image analysis highlights the flexibility of the OG domain, implicating its role in the varied enzyme conformations and in facilitating independent movements of the terminal domains. This may endorse recognition, binding, and processing of substrates, ligands, as well as receptors and marks this domain as an additional target for the design of selective regulators.