Phosphatidylinositol-4,5-bisphosphate mediates the interaction of syndecan-4 with protein kinase C

Recent studies have demonstrated that the cytoplasmic tail of syndecan-4, a widely expressed transmembrane proteoglycan, can activate protein kinase Calpha in vitro, in combination with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)). Syndecan-4 is involved in growth factor binding as well as in adhesion to extracellular matrix proteins, while PI-4,5-P(2) synthesis is modulated by growth factor and adhesion-generated signaling. The cooperative activation of PKCalpha by the proteoglycan and the phosphatidylinositol may constitute, therefore, an essential part of the cell's response to these extracellular signals. To characterize the activation mechanism of PKCalpha, we addressed here the nature of the interplay between syndecan-4, PI-4,5-P(2), and PKCalpha by measuring their mutual binding affinities and the specificity of their interactions. We found that the cytoplasmic tail of syndecan-4 is unlikely to bind directly to PKCalpha, and that this interaction critically depends on PI-4,5-P(2). The PI-4,5-P(2) specificity of the activation of PKCalpha is conferred by the cytoplasmic tail of syndecan-4, which has higher binding affinity for this phosphatidylinositol over phosphatidylinositol-3,4-bisphosphate and the -3,4,5-trisphospate. The activation is specific to PKCalpha and does not encompass the novel protein kinase C delta isoenzyme.     

Bacteria-Host Cell Interaction Mediated by Cellular Cholesterol/Glycolipid-Enriched Microdomains

Gram negative bacterial infection is a leading cause of fatality and is attributed, at least in part, to the bacteria's capacity to persist in the host in spite of appropriate antibiotic therapy. It has been suggested that bacteria evade antibiotics by hiding within host cells. We sought to investigate this important aspect of infections in mast cells, which are inflammatory cells found in close proximity to the host-environment interface and which have recently been reported to play a crucial role in the early innate immune response to bacteria. We examined mast cell interactions with FimH-expressing E. coli, one of the major opportunistic pathogens of humans. We determined that in serum free conditions, these bacteria were able to trigger mast cell uptake without loss of bacterial viability. CD48, a mannose containing GPI (glycosylphosphatidylinositol)-linked molecule was found to be the receptor of FimH-expressing E. coli in mouse mast cells. We found that the internalization via CD48 was blocked by filipin, a cholesterol binding drug known to disrupt cholesterol/glycolipid-enriched microdomains and the bacteria-encasing vacuoles were rich in cholesterol inside cells. Interestingly, we found that mast cells subsequently expelled majority of the intracellular bacteria in 24 hours. This expulsion process was blocked by lovastatin/cyclodextrin treatment, which is known to inhibit cellular trafficking of cholesterol/glycolipid-enriched microdomains. Thus, the bacterial entry into and expulsion from mast cells were critically dependent on cholesterol/glycolipid-enriched microdomains, which represents a novel mode of tussle between the pathogen and the mast cell occurring in opsonin deficient sites in the body or even at other sites in naive or immunocompromised hosts which have low systemic levels of E. coli specific antibody.     

Inhibition of Photosynthesis by Shift in the Balance of Excitation Energy Distribution Between Photosystems in Dithiothreitol Treated Soybean Leaves

Chlorophyll fluorescence kinetics was used to investigate the effect of 1,4-dithiothreitol (DTT) on the distribution of excitation energy between photosystem 1 (PS1) and photosystem 2 (PS2) in soybean leaves under high irradiance (HI). The maximum PS2 quantum yield (F_v/F_m) was hardly affected by the presence of DTT, however, photon-saturated photosynthesis was depressed distinctly. Photochemical efficiency of open PS2 reaction centres during irradiation (F_v/F_m) was enhanced by about 30–40 % by DTT treatment, whereas photochemical quenching (q_P) was depressed by about 40 % under HI. DTT treatment caused a 30 % decrease in allocation of excitation energy to PS1 under HI and a 20 % increase to PS2. An obvious shift in the balance of excitation energy distribution between photosystems was observed in DTT-treated leaves. Though high excitation pressure (1 - q_P) resulted from DTT treatment, non-photochemical quenching (q_N) was lower. DTT completely inhibited the formation of zeaxanthin and also distinctly depressed the state transition (q_T). The shift in the balance of excitation distribution between the two photosystems induced by DTT was mainly due to the enhancement of excitation energy capture by PS2 antenna and the inhibition of state transition. It might be the shift in the balance between the two photosystems that mainly induced the depression of photosynthesis. Thus, to keep high utilization efficiency of absorbed photon energy, it is necessary to maintain the balance of excitation distribution between PS2 and PS1.     

Protective effects of N-n-butyl haloperidol iodide on myocardial ischemia-reperfusion injury in rabbits

N-n-butyl haloperidol iodide (F2), a novel compound derived from haloperidol, was synthesized by our drugs research lab. The present study aims to evaluate the protective effects of F2 on myocardial ischemia-reperfusion injury in vivo, and to try to find the protective mechanism of F2. The animal model of myocardial ischemia-reperfusion injury was established by ligaturing rabbit's left ventricular branch of coronary artery for 40 min and removing the ligation later to reperfuse for 40 min. Different doses of F2 were intravenously injected before the onset of ischemia. The changes of hemodynamics were recorded during the experiment, and the activities of superoxide dismutase (SOD), creatine kinase (CK), Ca2+-ATPase, Na+,K+-ATPase and the level of malondialdehyde (MDA) of myocardial tissue were detected after reperfusion. Administration of F2 could dose-dependently ameliorate the hemodynamics of ischemia-reperfusion injured myocardium. During the course of reperfusion, MAP, LVSP, +/-dP/dt(max) in all F2 groups were obviously higher than those in the ischemia-reperfusion control group, and LVEDP were lower. F2 could also reduce the production of MDA, and maintain the activities of SOD, Ca2+-ATPase, Na+,K+-ATPase, and minimize the leakage of CK out of myocardial cells in a dose-dependent manner. These results suggested that F2 had apparent protective effects against myocardial ischemia-reperfusion injury.     

Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.     

Double-strand hydrolysis of DNA by a magnesium(II) complex with diethylenetriamine

The development of artificial nucleases that hydrolyze DNA or RNA is of great interest in molecular biology, biotechnology, and medicine. We now report that a magnesium(II) complex of diethylenetriamine (Mg-dien) can effectively promote the double-stranded cleavage of plasmid DNA and the dideoxynucleotide dApdA under physiological conditions of pH and temperature. Experiments performed in the presence of hydrogen peroxide, radical scavengers, or under rigorously anaerobic conditions indicate that DNA cleavage mediated by Mg-dien occurs via a hydrolytic path. Mg-dien efficiently hydrolyzes supercoiled pBR322 DNA and the pseudo-first-order rate constant at 37 degrees C and pH 8.0 is estimated to be 1.60 h(-1). The dinucleotide dApdA hydrolysis, with Mg-dien at 170 microM, shows a rate enhancement factor of ca. 5 x 10(8). 1H and 31P(1H) NMR studies show that Mg-dien effectively hydrolyzes 5'-dAMP to give deoxyadenosine and inorganic phosphate. While Mg2+ has been found at the catalytic sites of many natural nucleases, Mg-dien appears to be the first synthetic Mg2+-containing system capable of hydrolyzing dideoxynucleotides and DNA and thus may provide a simple model system to assist mechanistic studies of naturally occurring nucleases.     

Biological activity and conformational analysis of C20 and C14 epimers of CD-ring modified trans-decalin 1alpha,25-dihydroxyvitamin D analogs

In the context of our ongoing study of vitamin D structure-function relationships and in an attempt to obtain a better dissociation of their prodifferentiating (HL-60) and/or antiproliferative (MCF-7) activities and their calcemic activity, further 20-epi and 14-epi modifications were made to three trans-decalin CD-ring analogs of 1,25-dihydroxyvitamin D(3), the hormonally active metabolite of vitamin D(3), possessing a natural 20R side chain and featuring additional structural modifications in the seco-B-ring and in the A-ring. Following a previously observed trend and in agreement with the conformational analysis results, all three 20-epi derivatives show substantially lower biological activities, opposite to what is usually observed for analogs having the natural CD-ring. The 14-epi modification (cis-decalins) has little effect on the biological activity of the ynediene type and the saturated derivative, but results in an approximate 10-fold reduction in activity of the previtamin derivative. No better dissociation of the prodifferentiating and/or antiproliferative activities and the calcemic activity was achieved.