Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effects of muscle-conditioned medium

The enzymatic machinery for neurotransmitter synthesis and breakdown have been compared in sister cultures of newborn rat sympathetic neurons grown for 12-28 days either in the presence (CM+ cultures) or in the absence (CM- cultures) of a culture medium conditioned by rat skeletal muscle cells. Neuron numbers, total protein, and lactate dehydrogenase activities were identical in CM+ and CM- cultures. Choline acetyltransferase activity was 27- to 100-fold higher in homogenates of CM+ than CM- cultures, whereas acetylcholinesterase activity was 2.5-fold lower. The activities of tyrosine hydroxylase (TOH), DOPA decarboxylase, and dopamine beta-hydroxylase were all about twofold lower in homogenates from CM+ cultures. All these effects were also observed in homogenates of sympathetic neuron cultures grown with and without a macromolecular factor partially purified from CM (Weber, J. (1981). Biol. Chem. 256, 3447-3453.). Experiments of mixing homogenates from CM+ and CM- cultures suggested that the differences in each of the enzyme activities did not result from differences in the concentrations of hypothetical reversible enzyme activators and/or inhibitors. In addition, the deficit in TOH activity in CM+ cultures resulted from a decrease in the enzymatic Vmax with no significant variation in the apparent Km's for the substrate and the cofactor. An identical decrease in the Vmax was observed if TOH was assayed under phosphorylating or nonphosphorylating conditions, suggesting that this decrease did not result from differences in the state of enzyme phosphorylation. Immunoprecipitation curves of TOH activity by an anti-TOH antiserum were parallel when performed on homogenates from CM+ and CM- cultures, suggesting a difference in the number of enzyme molecules without detectable alteration of their kinetic properties.     

Selective binding of lectins to embryonic chicken vasculature.

Chicken embryos are an excellent model system for studies related to vascular morphogenesis. Development in ovo allows manipulations otherwise difficult in mammals, and the use of chicken-quail chimeras offers an additional advantage to this experimental system. Furthermore, the chicken chorioallantoic membrane has been extensively used for in vivo assays of angiogenesis. Surprisingly, few markers are available for a comprehensive visualization of the vasculature. Here we report the use of lectins for identification of embryonic chicken blood vessels. Nine lectins were evaluated using intravascular perfusion and directly on sections. Our results indicate that Lens culinaris agglutinin, concanavalin A, and wheat germ agglutinin can be used effectively for visualization of vessels of early chicken embryos (E2.5-E4). At later developmental stages, Lens culinaris agglutinin is a better choice because it displays equal affinity for the endothelia of arteries, veins, and capillaries. The findings presented here expand our understanding of lectin specificity in the endothelium of avian species and provide information as to the use of these reagents to obtain comprehensive labeling of the embryonic and chorioallantoic membrane vasculature.     

ARF- and coatomer-mediated peroxisomal vesiculation

The authors characterized on a molecular level the clofibrate-inducible 26-kDa integral peroxisomal membrane protein (Pmp26p, Pex11-1p) of rat liver. By screening cDNA databases with the obtained Pex11-1p-cDNA, a second homologous cDNA was identified that codes for a polypeptide with slightly larger molecular mass than Pex11-1p. The authors call this polypeptide Pex11-2p. Studies on the topology of Pex11-1p revealed two transmembrane domains with the N- and C-terminus facing the cytoplasm. The C-terminal tail of Pex11-1p ends in a consensus dilysine motif of the type-KXKXX-COOH, which is known to be involved in the ADP-ribosylation factor (ARF)1-coat protein (COP) I coat (ARF)1-dependent membrane recruitment to Golgi membranes. Studies with isolated peroxisomes incubated in the presence of cytosol, adenosine triphosphate and GTPγS, indeed, provided evidence for specific binding of ARF and coatomer to peroxisomes. Expression of Pex11-1p in Chinese hamster ovary (CHO) wild-type cells led to a twofold increase in the number of peroxisomes, but expression in a temperature-sensitive CHO mutant, defective in coatomer, induced elongation and tubulation of peroxisomal structures, rather than numerical proliferation. The obtained results for the first time offer a mechanism explaining Pex11-1p-, as well as ARF- and coatomer-mediated peroxisomal vesiculation. Two models are presented that may explain how these observations fit in with peroxisome biogenesis.     

Shelterin dysfunction and p16(INK4a)-mediated growth inhibition in HIV-1-specific CD8 T cells

HIV-1-specific cytotoxic T cell responses are expanded during advanced HIV-1 infection but seem unable to effectively protect the host against disease progression. These cells are able to produce gamma interferon and remain metabolically active but have defective proliferative activities, shortened telomeric DNA, and other signs of accelerated aging. To investigate the molecular mechanisms underlying the premature senescence of HIV-1-specific T cells, we focused here on the expression and function of a group of six nucleoproteins that are responsible for protecting and maintaining the structural integrity of telomeric DNA and are commonly referred to as "shelterin." We show that in progressive HIV-1 infection, the two major shelterin components TRF2 and TPP1 are selectively reduced in HIV-1-specific CD8 T cells, but not in T cells recognizing alternative viral species. This coincided with increased recruitment of 53BP1, a prominent DNA damage response factor, to telomeric DNA sites and was associated with elevated expression of the tumor suppressor p16(INK4a), which causes cellular growth inhibition in response to structural DNA damage. Notably, defective shelterin function and upregulation of p16(INK4a) remained unaffected by experimental blockade of PD-1, indicating a possibly irreversible structural defect in HIV-1-specific CD8 T cells in progressors that cannot be overcome by manipulation of inhibitory cell-signaling pathways. These data suggest that shelterin dysfunction and ensuing upregulation of the tumor suppressor p16(INK4a) promote accelerated aging of HIV-1-specific T cells during progressive HIV-1 infection.     

Symmetric dimers of ent-kaurane diterpenoids with cytotoxic activity from Croton tonkinensis

Breast cancer is the most common malignant tumor in women these days accounting for approximately 24% of all cancer. During our screening program searching for cytotoxic materials from natural products, two new symmetric dimers of ent-kaurane diterpenoid, crotonkinensins C (1) and D (2), with connectivity at C-17 were isolated from the leaves of the Vietnamese endemic medicinal plant Croton tonkinensis. Their structures were determined on the basis of physicochemical and spectroscopic data. Compound 2 showed a potent cytotoxic activity against MCF-7, tamoxifen-resistant MCF-7 (MCF-7/TAMR), adriamycin-resistant MCF-7 (MCF-7/ADR), and MDA-MB-231 breast cancer cell lines.     

Synthesis and anti Methicillin resistant Staphylococcus aureus activity of substituted chalcones alone and in combination with non-beta-lactam antibiotics

A total of 30 chalcone analogues was synthesized via a base catalyzed Claisen Schmidt condensation and screened for their in vitro antibacterial activity against Methicillin-sensitive Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) alone or in combination with non beta-lactam antibiotics namely ciprofloxacin, chloramphenicol, erythromycin, vancomycin, doxycycline and gentamicin. In the checkerboard technique, fractional inhibitory concentration indices (FICI) show that the following combinations like ciprofloxacin with 25 (4'-bromo-2-hydroxychalcone); doxycycline with 21 (4-hydroxychalcone); doxycycline with 25; and doxycycline with 4 (2',2-dihydroxychalcone) were synergistic against MRSA. In term SAR study, the relationship between chalcone structure and their antibacterial activity against S. aureus and synergy with tested antibiotics were discussed. Possible mechanisms for antibacterial activity of chalcones alone as well as the synergistic effect in combinations were proposed by molecular modeling studies, respectively. Combinations of chalcones with conventional antibiotics could be an effective alternative in the treatment of infection caused by MRSA.     

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.     

Supported bilayer lipid membranes modified with a phosphate ionophore

This article reports the electrical responses of a phosphate ionophore, the cyclic polyamine 3-decyl-1,5,8-triazacyclodecane-2,4-dione (N₃-cyclic amine) incorporated into metal supported bilayer lipid membranes (s-BLM). Teflon coated silver wire was used as a support. In a potentiometric mode, the ionophore had a response that was linearly related to the logarithm of HPO₄²⁻ concentration and was also dependant on pH. Selectivity coefficients for other anions compared to HPO₄²⁻ ions, determined by the separate solution method, fell within the range 1.73 × 10⁻⁴ to 6.38 × 10⁻².