Biomedical subjects
Publications and source records attributed to Y B Kim.
The crystal structure of N-[3-(3,4-dimethylphenyl)propyl] (4-hydroxy-3-methoxyphenyl)acetamide, C20H25NO3, was determined to a final R value of 0.047. The vanilloid, amide and dimethylphenyl groups of the compound are nearly perpendicular with respect to one another. The overall conformation in the crystalline state is somewhat different from that of other capsaicinoids. The molecules are stabilized by intermolecular hydrogen bonds.
G7 and PNK-E mAbs recognize distinct porcine NK cell and granulocyte function-associated molecules that enhance and induce a significant cytolytic response against tumor cell targets through a mechanism of re-directed cytotoxicity. The present study shows that the G7 and PNK-E molecules are present on the surface of porcine neutrophils, monocytes, and pulmonary alveolar macrophages as a physically and functionally associated cytolytic trigger molecular complex. Two-color flow cytometric analysis demonstrates that most, if not all, neutrophils are G7 and PNK-E antigen positive. In contrast, monocytes and PAM contain both G7 and PNK-E positive as well as G7 positive and PNK-E negative subpopulations. mAb binding competition experiments indicate that pretreatment of phagocytes with G7 mAb can block subsequent binding by PNK-E mAb, suggesting that these antigens are physically associated on the surface of porcine phagocytes. Fluorescent co-capping experiments utilizing G7 and PNK-E mAbs clearly demonstrate a physical association between G7 and PNK-E antigens present on the surface of porcine neutrophils. Pretreatment of phagocytes with F(ab')2 fragments of G7 and PNK-E mAbs shows that F(ab')2 G7 mAb blocks subsequent induction of phagocyte-mediated tumor cell cytotoxicity by whole PNK-E mAb but pretreatment with F(ab')2 PNK-E does not block subsequent induction of phagocyte-mediated tumor cell cytotoxicity by whole G7 mAb. In addition, pretreatment of neutrophils and mononuclear phagocytes with F(ab')2 fragments of G7 mAb blocks subsequent whole PNK-E mAb-dependent activation of a phagocytic cell intracellular oxidative burst response but pretreatment with F(ab')2 PNK-E does not block subsequent G7 mAb-dependent activation of a phagocytic cell intracellular oxidative burst response. These data reinforce a physical and functional association between the G7 and PNK-E molecules. Recent identification of the G7 antigen as the porcine homolog of Fc gamma RIII indicates that the G7 and PNK-E mAbs recognize a unique and previously uncharacterized Fc gamma R cytolytic trigger molecular complex present on the surface of porcine neutrophils, monocytes, and PAM.
Recently, vinyl sulfones have been observed to selectively inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is an important ATP-generating enzyme in glycolysis. The possibility of using GAPDH as a biochemical parameter of cytotoxicity by vinyl sulfones was investigated using mouse lymphocytes. Incubation of lymphocyte GAPDH with ethylvinyl sulfone resulted in a pseudo-first-order loss of enzyme activity. The exposure of lymphocytes to ethylvinyl sulfone resulted in the decrease of GAPDH activity followed by ATP depletion and cell death, which were both dependent on the concentration of ethylvinyl sulfone. A further study on the time-dependent change indicated that cell death was preceded by ATP loss. Compared to ethylvinyl sulfone, divinyl sulfone was more than 8 times more potent in causing either ATP depletion or cell death.
A possible participation of lysosomal hydrolases in the cytotoxicity of 2-chloroethylethyl sulfide in spleen lymphocytes was investigated using inhibitors of lysosomal phospholipases and proteases. Pepstatin (6 microM) and leupeptin (60 microM), inhibitors of lysosomal proteases, raised the viability of lymphocytes exposed to 2-chloroethylethyl sulfide from 63 to 87 and 88% of control, respectively. Serine protease inhibitors showed no significant effect on viability. Aminoglycoside inhibitors of lysosomal phospholipases were also found to prevent the decrease in viability of spleen lymphocytes exposed to 2-chloroethylethyl sulfide, and the effectiveness of these aminoglycosides (30 microM) was as follows: gentamicin > kanamycin > streptomycin, with viability increased to 89, 79 and 67%, respectively. In contrast to a co-operative action between leupeptin and gentamicin, the protection by pepstatin was reduced in the presence of gentamicin. Moreover, the order of the aminoglycosides in terms of the extent to which they antagonized the protective action of pepstatin was the same as their order of efficacy in preventing the cytotoxicity of CEES. It is suggested that inhibitors of lysosomal hydrolases reduce the cytotoxicity of 2-chloroethylethyl sulfide, presumably through lysosomal stabilization in spleen lymphocytes.
A hyperthermophilic archaebacterium was isolated from a deep-sea black smoker chimney (depth, 760 m) at the Minami-ensei Knoll (28 degrees 23'N, 127 degrees 38'E). The strain, designated DT1331, was a coccoid shaped bacterium about 0.5 to 1.0 microns in diameter. The cells were surrounded by a cell envelope. The temperature for growth was between 55 degrees C and 93 degrees C with an optimum 80 degrees C. The growth occurred from pH 4.5 to 8.5 and the optimum pH was 6.0. DT1331 required 1% to 5% NaCl for growth and cell lysis was observed below 1% NaCl concentration. The strain was an anaerobic chemoorganotroph requiring elemental sulfur obligately. Organic substrates used included tryptone, peptone, soytone, casein, gelatin, and yeast extract. Under the optimal conditions, DT1331 had a generation time of 50 min and could reach densities of about 1.5 x 10(8) cells/ml. DT1331 was resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, streptomycin, and tetracycline, which was one of the common characteristics of archaebacteria. The G+C content of DT1331 was 52.3 mol%. Analysis of the 16S rRNA gene by restriction enzymes coincided with those of Thermococcus celer, indicating that this strain belonged to the genus Thermococcus.
A 32-year-old male patient, diagnosed as a hepatic solitary angiomyolipoma post-operatively, is reported. The tumor was well definedly inhomogenous fat density mass on ultrasonography, computerized tomography (CT) and magnetic resonance imaging (MRI). The lesion was hypervascular on arteriography. The diagnosis was confirmed by an extended right lobectomy and histological examination.
The monoclonal antibodies G7 and PNK-E demonstrate potent enhancement/induction of porcine natural killer and polymorphonuclear leukocyte (PMN)-mediated tumor cell cytotoxicity and have been documented to exist as a molecular complex on the surface of porcine peripheral blood lymphocytes and PMN. This manuscript describes the biochemical characterization of the G7 molecule, recently identified as a porcine homologue of human Fc gamma RIIIA alpha, isolated from porcine leukocytes. Complete deglycosylation of the 43-kDa G7 molecule using N-glycanase reveals two polypeptides of 27 and 31 kDa in the majority of samples. V8 protease peptide mapping reveals structural similarities between the polypeptides and indicates the two polypeptides representing G7 isoforms. The previously described association of PNK-E with G7 and the coexpression of multiple G7 isoforms on porcine leukocytes suggest the presence of a novel Fc gamma RIII alpha molecular complex in the porcine system.
The G7 mAb binds a previously unidentified cytolytic trigger molecule on porcine NK cells and polymorphonuclear cells (PMN). This mAb has been shown to almost completely block PBL-mediated Ab-dependent cellular cytotoxicity and inhibit PMN-mediated Ab-dependent cellular cytotoxicity by approximately one-half, indicating a close functional association of the G7 molecule (to which the G7 mAb binds) with Fc gamma R function. This study was designed to identify the G7 molecule through molecular cloning of its cDNAs. The G7 protein was purified and peptide fragments subjected to amino acid sequencing. This information was then used in the PCR amplification of three cDNA fragments encoding a full-length G7 polypeptide. Amino acid sequences corresponding to peptides derived from G7 are observed within the deduced amino acid sequence of the individual clones confirming the specificity of G7 purification and PCRs. The specificity is also confirmed by reactivity of an antiserum raised against a synthetic G7 peptide to immunoprecipitated G7. Northern blot analysis indicates that a 0.9-kb G7 mRNA is expressed in porcine PMN, PBMC, macrophage, spleen, and lymph nodes but not in thymus or liver. Nucleotide and deduced amino acid sequence analyses indicate that G7 represents a porcine homologue to human Fc gamma RIIIA alpha. This is especially important in that G7 has been previously demonstrated to exist in a molecular complex with a novel molecule, PNK-E. The data suggests the presence of a novel Fc gamma RIII alpha molecular complex (G7/PNK-E) in the porcine system.
Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites.
Investigators from eight laboratories analyzed the reactivity of 22 monoclonal antibodies (mAb) against porcine myeloid cells. Based on binding data, clustering analysis and inhibition studies, workshop mAb 74-22-15 (003) and 6F3 (007) were assigned a swine workshop cluster number 3 (SWC3). These mAb recognized macrophages and neutrophils; neutrophils; a monocyte/macrophage-specific mAb was not identified by this workshop.
Four monoclonal antibodies (mAb) from the myeloid subset panel of the First International Swine CD Workshop (74-22-15, DH59B, PM16-6, and MUC21A) were analyzed using competitive inhibition studies with anti-human CD14 (My4) and anti-human/anti-porcine CD18 (MHM23) on porcine alveolar macrophages. Results suggested that none of the mAb tested recognized CD14 or CD18 on porcine alveolar macrophages. Additionally, the cross-reactivity of My4 with porcine alveolar macrophages was established.
Five monoclonal antibodies (mAbs), PNK-I (W #037), H20A (W #077), MUC76A (W #078), MUC93A (W #079) and MHM23 (W #136) of CD11/18 panels reacted with 80-96% of porcine PBMC and PMN. Epitope mapping studies by competitive binding of mAb by flow cytometric analysis based on PNK-I as CD18 epitope defining antibody resulted H20A and MHM23 mAbs bind to the same shared epitope as PNK-I, but MUC76A and MUC93A mAbs were distinct from PNK-I. Thus, PNK-I, H20A and MHM23 were designated as CD18a mAbs.
Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system.
Hypoglycemia increases the vulnerability of the perinatal brain to asphyxia, but it is not known if hypoglycemia-induced changes in cerebral hemodynamics and vascular reactivity underlie this vulnerability. This study tested the hypothesis that hypoglycemia exacerbates postischemic hypoperfusion, and impairs postischemic CO2 reactivity. The authors also examined the hypothesis that postischemic hypoperfusion is associated with a reduction in the interstitial concentration of the vasodilator metabolite adenosine. Global cerebral ischemia of 10 minutes duration was induced in newborn pigs anesthetized with isoflurane by occlusion of subclavian and brachiocephalic arteries; cortical cerebral blood flow (CBF) and interstitial adenosine concentration were evaluated simultaneously using the combined hydrogen clearance/microdialysis technique. Hypoglycemia (blood glucose < 25 mg/dl) was induced by regular insulin (25 IU/kg) administered intravenously 2 hours prior to induction of ischemia. In the eight normoglycemic animals, baseline CBF was 38 +/- 4 ml/min/100 gm and baseline adenosine concentration was 1.2 +/- 0.1 microM; in the eight hypoglycemic animals, these values were 39% (p < 0.05) and 62% (p < 0.05) greater, respectively, under baseline conditions. At 1 hour of postischemic reperfusion in normoglycemic animals, CBF was reduced 39% relative to the preischemic baseline (p < 0.01), concomitant with a 27% reduction (p < 0.05) in adenosine concentration, suggesting that this lowered concentration may underlie delayed hypoperfusion. These postischemic reductions in CBF and interstitial adenosine concentration were significantly greater in hypoglycemic animals, with CBF and adenosine concentration reduced 70% (p < 0.001) and 71% (p < 0.01), respectively, relative to baseline. In nine animals preischemic reactivity to hypercapnia was unaffected by hypoglycemia. Postischemic hypercapnic reactivity was retained in the eight normoglycemic animals, but was attenuated 73% (p < 0.05) in hypoglycemic animals. Thus, in the newborn pig, hypoglycemia exacerbates postischemic cortical hypoperfusion and impairs postischemic cerebrovascular reactivity to hypercapnia.
Our previous experimental work with tissue plasminogen activator (TPA) suggested the possibility of the clearance of vitreous hemorrhage by repetitive injections of low-dose TPA. We therefore investigated in rabbits the effect of both repeated injections of TPA and the change of the integrity of the vitreous body on the clearance of vitreous hemorrhage. Vitreous hemorrhage was produced by intravitreal injection of 0.05 ml of autologous whole blood in the pigmented rabbit eyes with intact vitreous or gas-compressed vitreous. Three intravitreal injections of 3-g TPA (total dose of 9 micrograms), separated by 7-day intervals, were performed. The endpoint for vitreous hemorrhage clearance was defined as clear visualization of the posterior central retina of the rabbits. Regardless of whether gas compression vitrectomy was performed, repeated injections of low-dose TPA resulted in rapid clearance of fresh vitreous hemorrhage in approximately two to three weeks after the last TPA injection. No evidence of retinal toxicity was seen in all experimental groups. Repetitive injections of low-dose TPA may be effective in the treatment of fresh vitreous hemorrhage.
Expanded polytetrafluoroethylene(ePTFE) grafts 4mm long and 1mm in diameter were implanted into the iliac artery of 100-150g male rats using standard microvascular technique. Prior to clamp removal, the cremaster muscle was isolated as an island flap based on the iliac artery and observed using intravital fluorescence microscopy. Fields which contained a bifurcation of a first order arteriole(80-100 microns diameter) into second order arteriole(50-80 microns) were chosen for observation. Platelets were labeled in vivo with acridine red to visualize and quantify the aggregates. Images of microemboli were counted manually and the area was measured by computerized planimetry. Six control grafts were implanted with no further processing, six were irrigated with heparin, and six were coated with tridodecylmethylammonium chloride(TDMAC) and heparin. Most thrombi appeared within the first five minutes after implantation in all groups. The total number of emboli observed in the control group was 91 pr animal, in the heparin irrigation group it was 84, and in the TDMAC-heparin group it was 22. The total thrombus area observed per animal was 137,660 +/- 29,467 microns 2 in the control group, 79,040 +/- 10,893 microns 2 in the heparin irrigation group, and 17,498 +/- 6,059 microns 2 in the TDMAC-heparin group (p < .01 vs control or heparin irrigation group). With this results we could find that heparin irrigation and TDMAC-heparin coating appear to reduce the number, size, and total amount of microemboli generated by ePTFE graft implantation and apparent thromboresistant property of TDMAC-heparin coating may have widespread application in many clinical and research areas and this experimental model can be used for evaluation of other graft matrials.
The large conductance Ca2+ activated K+ channel (BK channel) has been considered to play an important role in the excitability and contractility of vascular smooth muscle cells. Activation of the BK channel causes the hyperpolarization and relaxation of vascular smooth muscle cells. It has been reported that fatty acids can affect the BK channel activity and its concentration is increased significantly during myocardial ischemia. These reports suggest that fatty acids may contribute to the ischemic coronary vasodilation by increasing the BK channel activity. However, the underlying mechanism of fatty acid-induced activation of the BK channel is still uncertain. In the present study, we measured the effect of fatty acids on the BK channel activity in rabbit coronary smooth muscle cells by using patch clamp method and also examined its underlying mechanism. Arachidonic acid (AA) dissolved in DMSO activated the BK channel in a dose-dependent manner (from 0.5 to 10 microM), and DMSO (0.1%) alone had no effect on the activity of the BK channel. Arachidonic acid activated BK channels in both cell-attached and inside-out patches, but the onset and recovery of this effect were slower in the cell-attached patch configuration. The BK channel activity was also increased by other fatty acids, including myristic acid, linoleic acid, palmitoleic acid and palmitic acid. Long chain fatty acids were more effective than short chain fatty acids (myristic acid), and there was no statistical difference between the effect of saturated (palmitic acid) and unsaturated fatty acids (palmitoleic acid) on the BK channel activity. The concentration of Ca2+ and Mg2+ in the bathing solution had no appreciable effects on the AA-induced increase of BK channel activity. From the above results, it may be concluded that fatty acids directly increase the BK channel activity and may contribute to the ischemic coronary vasodilatation in rabbit coronary smooth muscle cells.
PNK-E and G7 mAb bind to distinct porcine granulocyte function-associated molecules and induce granulocyte-mediated cytotoxicity against tumor targets. PNK-E mAb binds to a 205-kDa molecule that reduces to a dispersed band of 50 kDa on SDS-PAGE analysis. Previous work demonstrates that G7 mAb immunoprecipitates a molecule that appears as a heterodispersed 40-kDa band under both reducing and nonreducing conditions on SDS-PAGE analysis. Whole but not F(ab')2 PNK-E and G7 mAb induce dose-dependent porcine granulocyte-mediated lysis of FcR+ but not FcR- targets, suggesting a redirected cytotoxicity mechanism of granulocyte-mediated killing. Fresh porcine granulocytes also mediate significant levels of antibody-dependent cellular cytotoxicity (ADCC) against nucleated (SB) target cells. Neither whole nor F(ab')2 PNK-E mAb affects granulocyte-mediated ADCC against SB target cells. However, both whole and F(ab')2 G7 mAb inhibit granulocyte-mediated ADCC against SB targets by approximately 50%. Bound F(ab')2 G7 mAb inhibits PNK-E mAb-induced granulocyte-mediated cytotoxicity against K562 targets, but bound F(ab')2 PNK-E mAb does not inhibit G7 mAb-induced granulocyte-mediated cytotoxicity, suggesting a physical association between the PNK-E and G7 molecules on the surface of porcine granulocytes. PNK-E and G7 hybridoma cells are readily lysed by granulocyte effectors, further supporting that the PNK-E and G7 molecules are cytolytic trigger molecules on granulocytes. These data demonstrate that PNK-E and G7 mAb bind to distinct granulocyte lytic trigger molecules and induce potent granulocyte-mediated cytotoxicity against nucleated tumor targets through a mechanism of redirected cytotoxicity.