We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.
Calcium Channels ; Chromatography, High Pressure Liquid ; Crotalus* ; Electrophoresis, Polyacrylamide Gel ; Humans* ; Membranes ; Neutrophils ; Phosphodiesterase I ; Phospholipases A2 ; S Phase ; Venoms*

Skin is the outermost organ and acts as a barrier between the organism and environment. Skin protects the organism from environmental insults, such as chemicals, pathogens, and UV light. Much of the protective function of skin is dependent on the epidermis, a multi-layered epithelium that is composed of various cell types such as keratinocytes and melanocytes. Keratinocytes produce protective components through a sophisticated differentiation process. Disturbance of keratinocyte differentiation is related to several skin diseases such as psoriasis and atopic dermatitis. In this study, we prepared extract of combined medicinal plants (ECMP) consisting of Taraxacum platycarpum H. Dahlstedt, Heartleaf Houttuynia, Glycyrrhiza uralensis Fischer, and root bark of Ulmus davidiana. We demonstrated that ECMP enhanced keratinocyte differentiation and barrier functionality using an in vitro cell culture system and in vivo animal test. Treatment of cultured keratinocytes with ECMP resulted in induction of keratinocyte differentiation, as evidenced by increased differentiation markers such as involucrin, loricrin, and filaggrin. In line with these results, ECMP decreased proliferation of keratinocytes cultured in vitro. ECMP applied topically to tape-stripped mouse skins accelerated reduction of transepidermal water loss (TEWL), indicating fast recovery of barrier function. Immunohistochemistry showed that ECMP increased the filaggrin level in tape-stripped mouse skins. These results suggest that ECMP may be applicable for keratinocyte differentiation-related skin diseases.
Animals ; Antigens, Differentiation ; Cell Culture Techniques ; Dermatitis, Atopic ; Epidermis ; Epithelium ; Glycyrrhiza uralensis ; Houttuynia ; Immunohistochemistry ; Keratinocytes* ; Melanocytes ; Mice ; Plants, Medicinal* ; Psoriasis ; Skin ; Skin Diseases ; Taraxacum ; Ulmus ; Ultraviolet Rays

OBJECTIVETo explore the feasibility of applying autosomal single nucleotide polymorphisms (SNPs) on parentage testing.

METHODSAll SNP genotyping results of HapMap (r27) were downloaded from the website. With self-made computer programs, SNPs were extracted when their minor allele frequency (MAF) were ≥ 0.30 among all of the 11 HapMap populations. Ninety-six SNPs were chosen and integrated into the Illumina Goldengate bead arrays on the condition that no linkage disequilibrium was found between them. Three father-child-mother trios (9 samples in total) were tested with the arrays. Cumulative paternity index (CPI) was then calculated and compared with genotyping results using 15 short tandem repeats (STRs)(Identifiler(TM)).

RESULTSFamily 1 was found to have nine SNPs or seven STRs that did not conform to the Mendelian laws, Family 2 had 13 such SNPs or seven STRs, and Family 3 only had one such SNP but no STR. For Family 3, when all of the 96 SNPs were used in combine, the CPI was 1207, which had contrasted with the CPI by the 15 STRs, i.e., 355 869.

CONCLUSIONWhen applied to paternity testing, the paternity exclusion (PE) value for a SNP is usually less than 1/3 of that of a STR. The proportion of SNPs not comforming to the Mendelian laws for the tested SNPs may not be as high as that of inconsistent STRs over all tested STRs. Because of the low mutation rate of a SNP, the CPI will be greatly reduced even if one SNP did not conform to the Mendelian laws. Therefore, highly accurate testing methods are required to reduce artificial errors when applying SNPs for paternity testing.

Fathers ; Female ; Genetic Testing ; methods ; Genotype ; HapMap Project ; Humans ; Male ; Mothers ; Paternity ; Polymorphism, Single Nucleotide ; genetics

We demonstrate that glycoprotein isolated from Dioscorea batatas (GDB) has immunostimulatory effects including macrophage activation. Analysis of infiltration of inflammatory cells into peritoneal cavity showed GDB treatment significantly increased the recruitment of macrophages, lymphocytes, neutrophils, and monocytes into the peritoneal cavity. Treatment of spleen cells isolated from C57BL/6 mice with GDB significantly increased the proliferation of B cells and T cells induced by LPS and ConA, respectively. Treatment with GDB significantly increased the cytolytic capacity of NK cells and macrophages against YAC-1 and B16 cells, respectively. In order to further confirm and investigate the mechanism of GDB on macrophage activation, we analyzed the effects of GDB on the cytokine expression including iNOS, IL-1beta, and TNF-alpha in mouse macrophage cell line, RAW 264.7 cells. RT-PCR and ELISA showed that GDB increased the expression of IL-1beta, and TNF-alpha, whereas iNOS was not induced by GDB. Collectively, this series of experiments indicates that GDB stimulates immune system including macrophage activation.
Animals ; B-Lymphocytes ; Cell Line ; Dioscorea ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; Immune System ; Killer Cells, Natural ; Lymphocytes ; Macrophage Activation ; Macrophages ; Mice ; Monocytes ; Neutrophils ; Peritoneal Cavity ; Spleen ; T-Lymphocytes ; Tumor Necrosis Factor-alpha

BACKGROUND: Caffeic acid phenethyl ester (CAPE) is an active component of propolis and is known to have anti-inflammatory properties. This study was performed to evaluate the effects of CAPE on lipopolysaccharide (LPS)-induced murine macrophage activation. METHODS: Raw 264.7 cells were incubated with varying concentrations of CAPE with or without LPS. The production of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and macrophage inflammatory protein-2 (MIP-2) and activation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun amino terminal kinases (JNK) and p38 were measured. RESULTS: CAPE inhibited the production of TNF-alpha, IL-1beta and MIP-2 and attenuated phosphorylation levels of ERK1/2 and p38, but not JNK in RAW264.7 cells stimulated with LPS. CONCLUSIONS: CAPE can attenuate LPS-induced macrophage responses and we suggest that these effects may play an important role in modulating macrophage-mediated inflammatory responses in vivo.
Caffeic Acids ; Cytokines ; Extracellular Signal-Regulated MAP Kinases ; Interleukins ; Macrophage Activation ; Macrophages ; Mitogen-Activated Protein Kinases ; Phenylethyl Alcohol ; Phosphorylation ; Phosphotransferases ; Propolis ; Tumor Necrosis Factor-alpha

PURPOSE: Ischemia and reperfusion (I/R) injury is a major cause of hepatic failure after liver surgery, but there is no direct method to monitor it in real-time (like an electrocardiogram in heart disease) during surgery. Recently we found the possible role of bioelectrical impedance (BEI) to monitor I/R injury in liver. But the mechanism responsible for ischemia-related BEI changes has not been clearly determined. METHODS: The authors used an LCR meter to quantify BEI changes at 0.12 KHz. Livers were subjected to 70% partial ischemia for 120 minutes, and ATP contents, cation changes in extracellular fluid (ECF; determined using an in vivo intracellular microdialysis technique), hepatocyte sizes, and histological changes were then examined. RESULTS: Liver tissue BEI was found to increase gradually during the first 60 minutes of ischemia and then tended to plateau. During the same period, intracellular ATP contents decreased to below 20% of the baseline level, [Na+] in ECF decreased from 150.4+/-3.8 to 97.8+/-10.6 mmol/L, and [K+] in ECF increased from 7.5+/-0.3 to 34.3+/-5.5 mmol/L during the first 60 minutes of ischemia. Hepatocyte diameter increased by ~20% during the first 60 minutes of ischemia. CONCLUSION: This study suggests that BEI changes during hepatic ischemia are probably caused by sodium and potassium concentration changes in the ECF due to reduced intracellular ATP contents.
Adenosine Triphosphate ; Electric Impedance ; Electrocardiography ; Extracellular Fluid ; Heart ; Hepatocytes ; Ischemia ; Liver ; Liver Failure ; Microdialysis ; Organothiophosphorus Compounds ; Potassium ; Reperfusion ; Sodium

PURPOSE: The aim of this study was to present our experience before establishing laparoscopic left lateral sectionectomy (LLLS) of the liver as a standard procedure, and to show efficacy of a totally LLLS compared to an open left lateral sectionectomy (OLLS). METHODS: We retrospectively analyzed and compared clinical outcomes (operation time, blood loss, hospital stay, complication rate, etc) for 29 patients who underwent LLLS and 27 patients who underwent OLLS between January, 2002 and December, 2009. To see the learning curve for LLLS, we arbitrarily divided the LLLSs we did into an early group (ELLLS) and a late group (LLLLS) based on when they were operated on relative to case number 14. RESULTS: Mean operative times for the ELLLS, LLLLS and OLLS groups were 269.7+/-102.6, 210.0+/-47.9 and 289.1+/-72.8 minutes, respectively. Mean operative time was significantly shorter (p<0.05) in the LLLLS than the OLLS group. Mean intra-operative blood loss was also less in the LLLLS group than the OLLS group (80.00+/-224.2 ml vs. 195.15+/-405.4 ml). Post-operative hospital stay was shorter in the LLLLS group than the OLLS group (9.9+/-4.0 versus 16.9+/-9.1, p=0.071). CONCLUSION: The totally LLLS is a safe, feasible treatment option that can be a standard procedure with better outcomes in selected patients after an initial learning curve.
Humans ; Laparoscopy ; Learning Curve ; Length of Stay ; Liver ; Operative Time ; Retrospective Studies

Ischemia and reperfusion (I/R) injury is a major cause of hepatic failure after liver surgery, but no method could monitor or predict it real-time during surgery. We measured bioelectrical impedance (BEI) and cell viability to assess the usefulness of BEI during I/R in rat liver. A 70% partial liver ischemia model was used. BEI was measured at various frequencies. Adenosine triphosphate (ATP) content, and palmitic acid oxidation rate were measured, and histological changes were observed in order to quantify liver cell viability. BEI changed significantly during ischemia at low frequency. In the ischemia group, BEI increased gradually during 60 min of ischemia and had a tendency to plateau thereafter. The ATP content decreased below 20% of the baseline level. In the I/R group, BEI recovered to near baseline level. After 24 hr of reperfusion, the ATP contents decreased to below 50% in 30, 60 and 120 min of ischemia and the palmitic acid metabolic rates decreased to 91%, 78%, and 74%, respectively, compared with normal liver. BEI may be a good tool for monitoring I/R during liver surgery. The liver is relatively tolerant to ischemia, however after reperfusion, liver cells may be damaged depending upon the duration of ischemia.
Adenosine Triphosphate/metabolism ; Animals ; *Cell Survival ; Electric Impedance ; Energy Metabolism ; Ischemia/*metabolism ; Liver/*metabolism/pathology ; Male ; Palmitates/metabolism ; Rats ; Rats, Sprague-Dawley ; *Reperfusion ; Reperfusion Injury/metabolism/pathology

To investigate the intestinal absorption of a fibrinolytic and proteolytic lumbrokinase extracted from Eisenia andrei, we used rat everted gut sacs and an in situ closed-loop recirculation method. We extracted lumbrokinase from Eisenia andrei, and then raised polyclonal antibody against lumbrokinase. Fibrinolytic activity and proteolytic activity in the serosal side of rat everted gut sacs incubated with lumbrokinase showed dose- and time-dependent patterns. Immunological results obtained by western blotting serosal side solution using rat everted gut sacs method showed that lumbrokinase proteins between 33.6 and 54.7 kDa are absorbed mostly by the intestinal epithelium. Furthermore, MALDI-TOF mass spectrometric analysis of plasma fractions obtained by in situ recirculation method confirmed that lumbrokinase F1 is absorbed into blood. These results support the notion that lumbrokinase can be absorbed from mucosal lumen into blood by oral administration.
Administration, Oral ; Animals ; Blotting, Western ; Endopeptidases ; Intestinal Absorption ; Intestinal Mucosa ; Oligochaeta ; Plasma ; Proteins ; Rats

OBJECTIVE: Aquaporin (AQP) 3 is a small integral membrane protein that functions as a facilitated transporter of water and glycerol. To elucidate a role of AQP3 in placenta, changes in amniotic fluid composition and fetal growth were investigated using AQP3 null mice. METHODS: Embryonic day 14,5 gestational sacs of wild-type and AQP3 kncok-out pregnant mice, thirty each, were used for this study. AQP3 localization and expression were assessed by immunohistochemistry and western blot. RESULTS: AQP3 was highly expressed in basolateral membrane of visceral yolk sac cells of fetal membrane and syncytiotrophoblast cells of labyrinthine placenta. In contrast, AQP1 was expressed in apical membrane of visceral yolk sac cells and endothelial cells lining vasculature. There was no significant difference in normal placentation and differentiation from trophoblast stem cells between wild type and AQP3 null mice. However, AQP3 null mice had increased amount of amniotic fluid per gram of body weight and decreased osmorality of amniotic fluid with low concentrations of ions and solutes in amniotic fluid. In addition, AQP3 null mice pups were smaller than CD1 wild type mice. CONCLUSION: AQP3 plays an important role in amniotic water balance and nutrient supply to developing fetus by facilitating transplacental transport of water and glycerol.
Amniotic Fluid ; Animals ; Blotting, Western ; Body Weight ; Endothelial Cells ; Extraembryonic Membranes ; Female ; Fetal Development ; Fetus ; Gestational Sac ; Glycerol ; Immunohistochemistry ; Ions ; Membrane Proteins ; Membranes ; Mice ; Placenta ; Placentation ; Stem Cells ; Trophoblasts ; Water ; Yolk Sac