Matrix metalloproteinase-2, membranous type 1 matrix metalloproteinase,and tissue inhibitor of metalloproteinase-2 expression in ectopic and eutopic endometrium

OBJECTIVE: To investigate expression of matrix metalloproteinase-2 (MMP-2), membranous type 1 matrix metalloproteinase (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2) in ectopic and eutopic endometrium from women with and without endometriosis throughout the menstrual cycle. DESIGN: Molecular studies in human tissue. SETTING: Reproductive immunology laboratory of a university medical center. PATIENT(S): Fifty-three premenopausal woman (23 with endometriosis and 30 without endometriosis) undergoing laparoscopic surgery. Endometrium and ectopic endometriosis tissue were obtained at the time of surgery. MAIN OUTCOME MEASURE(S): Messenger RNA and protein expression from eutopic and ectopic endometrium was analyzed by using quantitative competitive polymerase chain reaction, zymography, and Western blot assay. RESULT(S): Uterine endometrium from women with endometriosis expressed higher levels of MMP-2 and MT1-MMP and lower levels of TIMP-2 than did endometrium from normal women. CONCLUSION(S): Eutopic endometrium from patients with endometriosis may be more invasive and prone to peritoneal implantation because of greater expression of MMP-2 and MT1-MMP and lower expression of TIMP-2 messenger RNA, compared with endometrium from women without endometriosis. Thus, increased proteolytic activity may help to explain the invasive factors that result in endometriosis.     

A direct sandwich ELISA to detect antibodies against the C-terminal region of merozoite surface protein 1 could be a useful diagnostic method to identify Plasmodium vivax exposed persons

We expressed a C-terminal 108-aa region of the Plasmodium vivax merozoite surface protein 1 (PvMSP1c) excluding the C-end transmembrane region in Escherichia coli in order to evaluate the antibody level to MSP in Korean malaria patients. We optimized a direct sandwich enzyme-linked immunosorbent assay (ELISA) method to simultaneously determine the total antibody levels, including IgG and IgM, to PvMSP1c. If the cut-off for seropositivity was determined as the mean+3SD of the antibody levels of the negative control group, the antibody levels were positive in 99.5% of the patient group (sensitivity 199/200). The antibody levels were negative in 99.4% of the negative control group (specificity 504/507). The positive reactions in the negative control group came from non-specific reactions, as confirmed by a competition assay. This direct sandwich ELISA for PvMSP1c antibody could prove to be a useful tool for the diagnosis of malaria patients and for blood screening in blood banks.     

Altered modulation of soluble guanylate cyclase in lymphocytes from patients with liver disease

Studies of animal models suggest that the activation of soluble guanylate cyclase by nitric oxide is altered in liver disease. We studied 77 patients with liver disease and 17 controls, to investigate whether the activation of soluble guanylate cyclase is altered in lymphocytes from patients with liver disease. The basal content of guanosine 3',5'-cyclic monophosphate (cGMP) in lymphocytes was decreased both in patients with liver cirrhosis (by 52%) and in patients with chronic hepatitis (by 62%). Activation of soluble guanylate cyclase by nitric oxide was higher in lymphocytes from patients with cirrhosis (3100+/-1000% of basal) or with hepatitis (5200+/-2500% of basal) than in lymphocytes from controls (1200+/-500% of basal). cGMP in plasma was increased in patients with liver disease. Successful (but not unsuccessful) treatment with interferon of patients with hepatitis due to virus C reversed all the above alterations. Altered modulation of soluble guanylate cyclase by nitric oxide in liver disease may play a role in the hemodynamic alterations found in these patients.     

Novel mutation and multiple mutations found in the human butyrylcholinesterase gene

BACKGROUND: Mutations in human butyrylcholinesterase (BChE) are linked to low BChE activity and abnormal response to muscle relaxants. METHODS: Twenty Chinese patients with hepatic disease and low cholinesterase activity, and one Japanese patient and her mother were tested for BChE activity and BChE phenotype. The butyrylcholinesterase (BCHE gene) was amplified by polymerase chain reaction (PCR) and sequenced. Mutant BChE was expressed in 293 cells. RESULTS: A novel mutation was found in one Chinese patient at nucleotide 943, where A was changed to T (943 A-->T), causing substitution of threonine 315 by serine (T315S). The T315S mutant had half of the normal BChE activity. One Japanese patient with low BChE activity had three nucleotide substitutions, 355 C-->T, 988 T-->A, and 1615 G-->A. The amino acid substitutions were Q119stop, L330I, and A539T, respectively. The single mutant L330I had low BChE activity, but the double mutant L330I/A539T had normal activity. CONCLUSIONS: The L330I and the novel T315S mutation caused a decreased BChE activity. The T315S mutation is one of the first BChE mutations reported in the Chinese population. Multiple mutations in BChE may interact with each other in an intramolecular manner.     

Determination of sulpiride by capillary electrophoresis with end-column electrogenerated chemiluminescence detection

BACKGROUND: Capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)3(2+)]-electrogenerated chemiluminescence (ECL) detection is a promising method for clinical analysis. In this study, a method combining CE with Ru(bpy)3(2+) ECL (CE-ECL) detection that can be applied to amine-containing clinical species was developed, and the performance of CE-ECL as a quantitative method for determination of sulpiride in human plasma or urine was evaluated. METHODS: Sulpiride was separated by capillary zone electrophoresis in uncoated fused-silica capillaries [50 cm x 25 microm (i.d.)] filled with phosphate buffer (pH 8.0) and a driving voltage of +15 kV, with end-column Ru(bpy)3(2+) ECL detection. A platinum disc electrode was used as working electrode. Sulpiride in human plasma or urine samples (100 microL) was extracted by a double-step liquid-liquid extraction procedure, dried under nitrogen at 35 degrees C in a water bath, and reconstituted with 100 microL of filtered water. The extraction solvent was ethyl acetate-dichloromethane (5:1 by volume). RESULTS: Under optimum conditions (pH 8.0 phosphate buffer, injection for 6 s at 10 kV, and +1.2 V as detection potential), separation of sulpiride was accomplished within 4 min. The calibration curve was linear over a concentration range of 0.05-25.0 micromol/L, and the limit of detection was 2.9 x 10(-8) mol/L for sulpiride. Intra- and interday CVs for ECL intensities were <6%. Extraction recoveries of sulpiride were 95.6-101% with CVs of 2.9-6.0%. The method was clinically validated for patient plasma and urine samples. CONCLUSIONS: CE combined with Ru(bpy)3(2+) ECL is reproducible, precise, selective, and enables the analysis of sulpiride in human plasma and urine. It thus is of value for rapid and efficient analysis of amine-containing analytes of clinical interest.