The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling

Signals from transforming growth factor-beta (TGF-beta) ligands are transmitted within the cell by members of the Smad family, which can be grouped into three classes based on sequence similarities. Our previous identification of both class I and II Smads functioning in a single pathway in C. elegans, raised the issue of whether the requirement for Smads derived from different classes is a general feature of TGF-beta signaling. We report here the identification of a new Drosophila class II Smad, Medea, a close homolog of the human tumor-suppressor gene DPC4. Embryos from germline clones of both Medea and Mad (a class I Smad) are ventralized, as are embryos null for the TGF-beta-like ligand decapentaplegic (dpp). Loss of Medea also blocks dpp signaling during later development, suggesting that Medea, like Mad, is universally required for dpp signaling. Furthermore, we show that the necessity for these two closely related, non-redundant Smads, is due to their different signaling properties - upon activation of the Dpp pathway, Mad is required to actively translocate Medea into the nucleus. These results provide a paradigm for, and distinguish between, the requirement for class I and II Smads in Dpp/BMP signaling.     

Effect of tuftsin on human Kupffer cell

BACKGROUND/AIMS: Kupffer cells are the most important category of reticuloendothelial cells which are critical for host defense in the liver. We investigated the effects of tuftsin (Thr-Lys-Pro-Arg) on human Kupffer cells. METHODOLOGY: Human Kupffer cells were obtained from the livers of patients with colon cancer. Phagocytosis assay was done by microscopic counting of the number of Kupffer cells that engulfed fluorescent particle(s), and the number of the particles engulfed per Kupffer cell when Kupffer cells were incubated with and without tuftsin. Effect of tuftsin on the release of tumor necrosis factor from Kupffer cells was also studied. RESULTS: Phagocytosis was enhanced significantly by tuftsin. The greatest effect on percentage of phagocytic cells was observed at 1.0 microg/ml of tuftsin. The mean number of particles engulfed per Kupffer cell was also increased with tuftsin 1.0 microg/ml. Tumor necrosis factor release was also significantly increased; the greatest effect was observed at 1.0 microg/ml of tuftsin. CONCLUSIONS: Tuftsin enhances phagocytic activity and tumor necrosis factor release of human Kupffer cells, which are advantageous for host defense against invading microorganisms and tumor cells.     

An essential role for a small synaptic vesicle-associated phosphatidylinositol 4-kinase in neurotransmitter release

Glutamate release from nerve terminals is the consequence of Ca2+-triggered fusion of small synaptic vesicles with the presynaptic plasma membrane. ATP dependence of neurotransmitter release has been suggested to be founded, in part, on phosphorylation steps preceding membrane fusion. Here we present evidence for an essential role of phosphatidylinositol phosphorylation in stimulated release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes). Specifically, we show that a phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity resides on nerve terminal-derived small synaptic vesicles (SSVs) and that inhibition of the PtdIns 4-kinase activity in intact synaptosomes leads to attenuation of the evoked release of glutamate. The attenuation of transmitter release is reversible and correlates with respective changes in intrasynaptosomal PtdIns 4-kinase activity. Because only the Ca2+-dependent release of glutamate is affected, regulation appears to be at the level of exocytosis. Taken together, our data imply a mandatory role for PtdIns 4-kinase and phosphoinositide products in the regulated exocytosis of SSV in mammalian nerve terminals.     

Microvascular effects of atrial natriuretic peptide (ANP) in man: studies during high and low salt diet

OBJECTIVE: Infusion of ANP in anephric dogs causes a decrease in cardiac output and a rise in peripheral vascular resistance. This reduced cardiac output is possibly related to increased resistance to venous return generated in the microcirculation by venular constriction. The aim of the present study was to evaluate in healthy volunteers the effects of low-dose ANP infusion on both conjunctival and skin microcirculation during high or low salt diet. METHODS: ANP (7.5 ng/kg/min) and placebo were infused (i.v.) for 4 h, in random order on two separate days, in two groups of 10 healthy male volunteers each. One group was studied during high salt (ad libitum), and one group during low salt (55 mmol Na+/24 h) diet. Microvascular density and diameters of both conjunctiva and nailfold were studied using intravital videomicroscopy. Nailfold capillary red blood cell velocity (CBV) was studied using intravital videomicroscopy, and skin (thermoregulatory) blood flow (SBF) was studied using laser-Doppler fluximetry. RESULTS: In the high salt group ANP induced a 43% reduction in basal SBF as compared to an 18% reduction by placebo (P < 0.01). Parallel to SBF, ANP significantly reduced CBV (P < 0.02). Conjunctival capillary density decreased by 5% during ANP, while it increased by 28% during placebo (P < 0.05). No such effects of ANP were observed in the low salt group. Blood pressure and heart rate were not influenced by ANP infusion in neither group. CONCLUSION: Infusion of low doses of ANP into humans on an ad libitum salt diet results in vasoconstriction of the microcirculation, probably on the venular side. The lack of effect of ANP on the microcirculation during low salt diet may be related to a higher vascular tone prior to infusion.     

Patient tolerance of craniotomy performed with the patient under local anesthesia and monitored conscious sedation

PURPOSE: To determine whether preoperative aortoiliac arteriography can be replaced with noninvasive evaluation in the management of some patients with chronic lower extremity ischemia. METHODS: Preoperative evaluation was performed on 184 ischemic limbs (119 patients) over 19 months by means of aortoiliac arteriography with runoff and noninvasive studies, which included common femoral artery duplex scanning, waveform and acceleration time (normal <140 msec), and aortoiliac duplex scanning. An algorithm was proposed for combining indirect (common femoral artery evaluation) and direct (aortoiliac evaluation) noninvasive studies to decrease the need for aortoiliac arteriography when possible. RESULTS: Aortoiliac occlusive disease (> or =50% stenosis to occlusion) was present at arteriography in 48 limbs (30%), and there was no inflow disease in 114 (70%). Aortoiliac lesions were identified by means of noninvasive studies. The accuracies of femoral waveform, acceleration time, and aortoiliac duplex studies were 85%, 89% and 87%. The negative predictive values were 92%, 94% and 100%. The acceleration time results were not affected by runoff status but were significantly different for various categories of stenosis (p < 0.05). The algorithm was applied to the data obtained. When acceleration time and waveform were normal, 84 of 86 patients (98%) had no stenosis at arteriography. When aortoiliac duplex findings were normal, the arteriographic findings were normal in all examinations. CONCLUSION: A combination of indirect and direct noninvasive studies can be used reliably to rule out clinically significant inflow occlusive disease and allows selective use of aortoiliac arteriography in patients with lower extremity ischemia.     

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

Analysis of commercial mini-bus accidents

The major challenge in liquid sustained-release oral suspensions is to minimize drug diffusion into the suspending medium and to retain the original properties of the microparticles during storage. Diclofenac wax microspheres prepared by the hydrophobic congealable disperse phase method were formulated as a sustained release suspension and stored at three different temperatures (25, 37 and 45 degrees C) for 3 months, to evaluate the physical and chemical stability of the suspended microspheres. Suspensions of microspheres stored at ambient temperatures were both physically and chemically stable, but at higher temperatures, up to 45 degrees C, there was a decrease in drug release due to scaling and melting on the microsphere surface as observed by scanning electron microscopy. However, on prolonged storage, up to 90 days, especially at 45 degrees C, temperature became a dominant factor causing an increase in drug release. The suspension of diclofenac microspheres was chemically stable for 3 months, while the plain drug suspension exhibited slight degradation.     

Amino acids 430-570 in apolipoprotein B are critical for its binding to microsomal triglyceride transfer protein

Several studies have demonstrated protein-protein interactions between microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). However, the binding sites involved in these interactions have not been elucidated. To identify an MTP binding site in apoB, we have expressed several apoB sequences as fusion proteins with the eight-amino acid FLAG peptide. The chimeras were transiently expressed in COS cells, and conditioned media were used to study the binding of these sequences to either immobilized or soluble MTP. A polypeptide containing amino acids 270-570 (B:270-570), but not 1-300, bound to MTP. AGI-S17, an antagonist of apoB-MTP binding, inhibited the binding of B:270-570 to MTP but not to M2, a monoclonal antibody that recognizes the FLAG peptide. These data indicated that B:270-570 contains an MTP binding site. Next, sequences within 270-570 were subjected to C-terminal truncations at natural proline residues. B:270-509 bound less efficiently than B:270-570, whereas, B:270-430 and other shorter chimeras did not bind to MTP. Furthermore, truncations at amino acids 502 and 509 decreased MTP binding by 73 and 42%, respectively. These data indicate that B:430-570 in the alpha1-globular domain of apoB plays a crucial role in MTP binding and presumably in the initiation and maturation of apoB-containing lipoproteins.