Synergism through direct covalent bonding between agents: a strategy for rational design of chemotherapeutic combinations

Self-assembling chemotherapeutic agents are mixtures of relatively nontoxic precursors that can combine chemically under physiological conditions to form products with greater cytotoxic and/or antimicrobial activity than either of the precursors. Combinations that form products more rapidly in or near the target (tumor, pathogen, virally infected cell) than in normal tissues will exhibit target-selective synergism, thus exhibiting an antitarget selectivity that is greater than the selectivities of the product (e.g., a hydrazone) and of either precursor (e.g., a hydrazine derivative or ketone) used singly. This paper describes the target-selective cytotoxic synergism of a cationic aldehyde (A) and a cationic acylhydrazine (B) containing a triarylalkylphosphonium moiety against Ehrlich ascites carcinoma cells (ELA) in culture, in addition to reviewing previous work on self-assembling cytotoxins. The synergism between A and B is carcinoma selective when the ELA cells (the target) are compared to CV-1, an untransformed African green monkey kidney epithelial line. Like tetraphenylphosphonium and rhodamine 123, which are selectively concentrated in ELA cells relative to CV-1, A, B and the hydrazone C resulting from their reaction are lipophilic delocalized cations that selectively inhibit ELA growth relative to CV-1 growth. The hydrazone C is more growth inhibitory than either A or B for both cell lines. A combination of A with an unreactive analogue of B and a combination of B with an unreactive analogue of A did not synergistically inhibit ELA proliferation. The degree of synergism is greater against the ELA cells than against the CV-1 cells. These data, together with hydrazone formation kinetics, suggest that A and B are both concentrated together selectively inside the ELA due to the transmembrane potentials, reacting inside the ELA cells at a higher velocity than inside the CV-1 cells to form the more growth-inhibitory hydrazone C.     

Engineered lentivector targeting of dendritic cells for in vivo immunization

We report a method of inducing antigen production in dendritic cells by in vivo targeting with lentiviral vectors that specifically bind to the dendritic cell-surface protein DC-SIGN. To target dendritic cells, we enveloped the lentivector with a viral glycoprotein from Sindbis virus engineered to be DC-SIGN-specific. In vitro, this lentivector specifically transduced dendritic cells and induced dendritic cell maturation. A high frequency (up to 12%) of ovalbumin (OVA)-specific CD8(+) T cells and a significant antibody response were observed 2 weeks after injection of a targeted lentiviral vector encoding an OVA transgene into naive mice. This approach also protected against the growth of OVA-expressing E.G7 tumors and induced regression of established tumors. Thus, lentiviral vectors targeting dendritic cells provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens.     

Sexual dimorphism: can you smell the difference?

A powerful new technique for visualizing neurons in the fly brain has uncovered fine neuroanatomical differences between the olfactory circuitries of male and female Drosophila.     

Citrullination, a possible functional link between susceptibility genes and rheumatoid arthritis

Antibodies directed to citrullinated proteins (anti-cyclic citrullinated peptide) are highly specific for rheumatoid arthritis (RA). Recent data suggest that the antibodies may be involved in the disease process of RA and that several RA-associated genetic factors might be functionally linked to RA via modulation of the production of anti-cyclic citrullinated peptide antibodies or citrullinated antigens.     

Nuclear cloning,stem cells,and genomic reprogramming

The generation of adult animals by nuclear cloning from adult donor cells is extremely inefficient, with most clones dying soon after implantation. In contrast, cloning from embryonic stem cell donor nuclei is significanty more efficient than from adult donor cells. However, regardless of donor cell type, all clones that survive to birth and beyond suffer serious phenotypic and gene expression abnormalities. All available evidence is consistent with the notion that the anomalous phenotypes of cloned animals are caused by faulty epigenetic reprogramming of the donor nucleus. Faulty reprogramming appears to be caused by the cloning process itself as well as by the epigenetic state of the donor nucleus. In contrast to reproductive cloning, faulty reprogramming of the donor nucleus does not tend to interfere with the application of nuclear transfer technology for therapeutic purposes (therapeutic cloning).     

Differential effects of Parkin and its mutants on protein aggregation, the ubiquitin-proteasome system, and neuronal cell death in human neuroblastoma cells

Mutations in Parkin, an E3 ligase, which participates in the ubiquitin-proteasome system (UPS), cause juvenile onset Parkinson's disease (PD). Some mutants aggregate upon over-expression, but the effects of such aggregation on the UPS and neuronal survival have not been characterized. We show in this study that transient over-expression of wild type (WT) Parkin or various mutants in human neuroblastoma cells leads to localized accumulation of green fluorescent protein (GFP(u)), an artificial proteasomal substrate, indicative of UPS dysfunction. Parkin mutants, but not WT, aggregated, and GFP(u) and ubiquitin accumulated within such aggregates. Apoptotic death occurred only with mutant Parkin over-expression, and correlated with aggregation, but not GFP(u) accumulation. Enzymatic proteasomal activity was slightly increased with WT Parkin and decreased with mutant Parkin over-expression. This decrease was, at least in part, due to caspase activation. We conclude that mutant forms of Parkin can exert toxic effects on neuronal cells, possibly through their propensity to aggregate. Both WT and mutant forms can induce localized UPS dysfunction, likely through different mechanisms. This raises a note of caution regarding forced over-expression of Parkin as a neuroprotective strategy in PD or other neurodegenerative conditions and suggests a possible toxic gain of function for certain mutant forms of Parkin.     

Analysis of human tyrosyl-DNA phosphodiesterase I catalytic residues

Tyrosyl-DNA phosphodiesterase I (Tdp1) is involved in the repair of DNA lesions created by topoisomerase I in vivo. Tdp1 is a member of the phospholipase D (PLD) superfamily of enzymes and hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and free tyrosine in vitro. Here, we use synthetic 3'-(4-nitro)phenyl, 3'-(4-methyl)phenyl, and 3'-tyrosine phosphate oligonucleotides to study human Tdp1. Kinetic analysis of human Tdp1 (hTdp1) shows that the enzyme has nanomolar affinity for all three substrates and the overall in vitro reaction is diffusion-limited. Analysis of active-site mutants using these modified substrates demonstrates that hTdp1 uses an acid/base catalytic mechanism. The results show that histidine 493 serves as the general acid during the initial transesterification, in agreement with hypotheses based on previous crystal structure models. The results also argue that lysine 495 and asparagine 516 participate in the general acid reaction, and the analysis of crystal structures suggests that these residues may function in a proton relay. Together with previous crystal structure data, the new functional data provide a mechanistic understanding of the conserved histidine, lysine and asparagine residues found among all PLD family members.     

Structure–activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y₁ while uridine containing dinucleotides have some level of agonist response on P2Y₂ and P2Y₆. The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Diurnal changes in net uptake rate of nitrate are associated with changes in estimated export of carbohydrates to roots

The rate of NO3- uptake by soybean (Glycine max [L.] Merrill) roots generally declines during the night in association with progressive depletion of the nonstructural carbohydrate pool in the shoot as well as the concentration of carbohydrates in roots. To determine if NO3- uptake rate changes in response to variations in translocation rate of carbohydrates from shoot to roots per se or to carbohydrate status of the roots, the night period was interrupted with a low light level from incandescent lamps to alter the diurnal pattern of NO3- uptake by roots and export of carbohydrate from shoots of nonnodulated soybean. Depletion of NO3- from replenished, complete nutrient solutions containing 1 mM NO3- was measured by ion chromatography and rates of NO3- uptake were calculated. Changes in export of carbohydrates from shoot to roots during intervals of the night period were calculated as the differences between rates of disappearance in contents of nonstructural carbohydrates and their estimated rates of utilization in shoot respiration and growth. A positive, significant correlation occurred between changes in calculated rates of carbohydrate export from shoots and NO3- uptake rates. Conversely, there was no significant correlation between concentrations of nonstructural carbohydrates in roots and NO3- uptake rates. These results support the hypothesis that carbohydrate flux from shoot to roots has a direct role in regulation of nitrogen uptake by the whole plant.