Optimized N-phenyl-N'-(2-chloroethyl)ureas as potential antineoplastic agents: synthesis and growth inhibition activity

In our ongoing research program aimed at the optimization of microtubule-self-assembly disrupting agents, we have prepared three series of phenylurea analogues (CEU), derived from N-(3-ω-hydroxyalkyl or 4-ω-hydroxyalkyl or 3-ω-hydroxyalkynyl)-phenyl-N′-(2-chloroethyl)ureas. Most compounds exhibit potent growth inhibitory activity on human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7 tumor cell lines, with a GI₅₀ ranging from 250 nM to 8 μM. Among these new molecules, three CEUs exhibit GI₅₀ in the nanomolar range. They are more potent by approximately an order of magnitude than previously described CEU analogues. As such, they are attractive hit compounds for the development of potent new alkylating antitubulin drugs.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Modeling and analysis of calcium signaling events leading to long-term depression in cerebellar Purkinje cells

Modeling and simulation of the calcium signaling events that precede long-term depression of synaptic activity in cerebellar Purkinje cells are performed using the Virtual Cell biological modeling framework. It is found that the unusually high density and low sensitivity of inositol-1,4,5-trisphosphate receptors (IP3R) are critical to the ability of the cell to generate and localize a calcium spike in a single dendritic spine. The results also demonstrate the model's capability to simulate the supralinear calcium spike observed experimentally during coincident activation of the parallel and climbing fibers. The sensitivity of the calcium spikes to certain biological and geometrical effects is investigated as well as the mechanisms that underlie the cell's ability to generate the supralinear spike. The sensitivity of calcium release rates from the IP3R to calcium concentrations, as well as IP3 concentrations, allows the calcium spike to form. The diffusion barrier caused by the small radius of the spine neck is shown to be important, as a threshold radius is observed above which a spike cannot be formed. Additionally, the calcium buffer capacity and diffusion rates from the spine are found to be important parameters in shaping the calcium spike.     

Evolutionarily divergent extradiol dioxygenases possess higher specificities for polychlorinated biphenyl metabolites

The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorinated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THBDRW1). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the Kmapp values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorinated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophobic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHBDP6-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHBDP6-III than was DHB. Indeed, DHBDP6-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THBDRW1 had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl. Nonetheless, THBDRW1 had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (kcatapp < 0.001 s(-1)).     

Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.     

The prodrug activator EtaA from Mycobacterium tuberculosis is a Baeyer-Villiger monooxygenase

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also are converted. EtaA is also able to catalyze enantioselective sulfoxidation of methyl-p-tolylsulfide. Conversion of all of the identified substrates is relatively slow with typical k(cat) values of around 0.02 s(-1). The best substrate identified so far is phenylacetone (K(m) = 61 microM, k(cat) = 0.017 s(-1)). Redox monitoring of the flavin cofactor during turnover of phenylacetone indicates that a step in the reductive half-reaction is limiting the rate of catalysis. Intriguingly, EtaA activity could be increased by one order of magnitude by adding bovine serum albumin. This reactivity and substrate acceptance-profiling study provides valuable information concerning this newly identified prodrug activator from M. tuberculosis.     

Differential role of lipid rafts in the functions of CD4+ and CD8+ human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4+ and CD8+ T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4+ and CD8+ T cells. The defect in IL-2 production by CD4+ T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56(Lck) and LAT and their association/recruitment with the lipid raft fractions of CD4+ and CD8+ T cells. p56Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4+ T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4+ and CD8+ T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4+ and CD8+ T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.     

Complex mosaicism in sex reversed SRY+ male twins

Sex reversal is characterized by discordance between genetic and phenotypic sex. Most XX males result from an unequal interchange between X and Y chromosomes during paternal meiosis, therefore transferring SRY to the X chromosome, which explains the male development in the presence of an otherwise normal female karyotype. We present here the case of sex reversed SRY+ male twins with several cell lines. They consulted for infertility. The presence of SRY on an X chromosome was demonstrated by FISH. Their respective karyotypes were: 46,X,der(X)t(X;Y)(p22.3;p11.2)[249]/45,X [12]/45,der(X)t(X;Y)(p22.3;p11.2)[11]/47,XX,der(X)t(X;Y) (p22.3;p11.2)[1]/47,X,der(X)t(X;Y)(p22.3;p11.2)x2[1]/50, XX,der(X)t(X;Y)(p22.3;p11.2)x4[1]/46,XX[1] for the first twin (SH-1) and 46,X,der(X)t(X;Y)(p22.3;p11.2)[108]/45,X [3]/47,XX,der(X)t(X;Y)(p22.3;p11.2)[2]/45,der(X)t(X;Y) (p22.3;p11.2)[1]/47,X,der(X)t(X;Y)(p22.3;p11.2)x2[1] for the second twin (SH-2). There are three different types of XX males: 1) with normal genitalia, 2) with genital ambiguity, and 3) XX true hermaphrodites. The phenotype of the twins presented in this report is consistent with what is generally seen in XX SRY+ males: they have normal genitalia.     

A glutathione S-transferase catalyzes the dehalogenation of inhibitory metabolites of polychlorinated biphenyls

BphK is a glutathione S-transferase of unclear physiological function that occurs in some bacterial biphenyl catabolic (bph) pathways. We demonstrated that BphK of Burkholderia xenovorans strain LB400 catalyzes the dehalogenation of 3-chloro 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), compounds that are produced by the cometabolism of polychlorinated biphenyls (PCBs) by the bph pathway and that inhibit the pathway's hydrolase. A one-column protocol was developed to purify heterologously produced BphK. The purified enzyme had the greatest specificity for 3-Cl HOPDA (kcat/Km, approximately 10(4) M(-1) s(-1)), which it dechlorinated approximately 3 orders of magnitude more efficiently than 4-chlorobenzoate, a previously proposed substrate of BphK. The enzyme also catalyzed the dechlorination of 5-Cl HOPDA and 3,9,11-triCl HOPDA. By contrast, BphK did not detectably transform HOPDA, 4-Cl HOPDA, or chlorinated 2,3-dihydroxybiphenyls. The BphK-catalyzed dehalogenation proceeded via a ternary-complex mechanism and consumed 2 equivalents of glutathione (GSH) (Km for GSH in the presence of 3-Cl HOPDA, approximately 0.1 mM). A reaction mechanism consistent with the enzyme's specificity is proposed. The ability of BphK to dehalogenate inhibitory PCB metabolites supports the hypothesis that this enzyme was recruited to facilitate PCB degradation by the bph pathway.     

Modulation of GJA1 turnover and intercellular communication by proinflammatory cytokines in the anterior pituitary folliculostellate cell line TtT/GF

Our previous studies have advanced the idea that the folliculostellate cell GJA1 (gap junction membrane channel protein alpha1; previously known as connexin 43)-mediated gap junctions contribute to the establishment of an intercellular network that regulates the paracrine messages and the endocrine response within the anterior pituitary. The folliculostellate cells are targets for growth factors and cytokines that modulate hormone secretion. Proinflammatory cytokines modulate the cell-to-cell communication in many tissues of the body. The present study measured the effect of the proinflammatory cytokines tumor necrosis factor and interleukin-1 on the GJA1-mediated intercellular communication, specifically the expression, localization, degradation, and phosphorylation status of GJA1 in the folliculostellate cell line TtT/GF. The GJA1 localized to the plasma membrane and to minute cytoplasmic vesicles in the perinuclear area. Using different antibodies that recognize distinctly the nonphosphorylated from the phosphorylated forms of GJA1, we showed that nonphosphorylated GJA1 in Ser-368 (NP-GJA1) localized chiefly in the cytoplasm, whereas GJA1 phosphorylated in Ser-368 (P-GJA1) localized to the plasma membrane in controls. The cytokine treatment transiently increased 1) GJA1, NP-GJA1, and P-GJA1 levels; 2) NP-GJA1 and P-GJA1 degradation by both the lysosomal and proteasomal pathways; and 3) cell-to-cell communication in TtT/GF cells. The results suggest that the cytokine-evoked, transient enhancement of folliculostellate cell-mediated intercellular communication contributes to the coordination of the response among folliculostellate cells.