The association of VEGF-C expression with tumor lymphatic vessel density and lymph node metastasis in patients with gastric cancer and gastrointestinal stromal tumor.

BACKGROUND/AIMS: Although tumors metastasize to lymph nodes via the lymphatics, the importance of vascular endothelial growth factor-C (VEGF-C) expression in mediating the process has not been well elucidated. We investigated the correlation between VEGF-C expression and lymphatic vessel density (LVD) and node metastasis in cases with gastric cancer and gastrointestinal stromal tumor (GIST). METHODOLOGY: Immunohistochemistry, VEGF-C expression and LVD were performed in 41 patients with gastric cancer invading the muscularis propria and 19 patients with GIST. The clinicopathological features of these cases were compared. RESULTS: In gastric cancer, VEGF-C expression was significantly associated with tumor LVD and lymph node metastasis. In GIST, none of these patients had lymph node metastasis and VEGF-C expression was not detected. The LVD was significantly higher in the cases with gastric cancer than in those with GIST. In gastric cancer, LVD was increased more in patients with positive lymph nodes than in those with negative lymph nodes. CONCLUSIONS: These results indicate that the expression of VEGF-C is associated with tumor LVD and lymph node metastasis, suggesting that VEGF-C plays a critical role in node metastasis via lymphangiogenesis. The clinical observation that GIST rarely metastasizes to the lymph nodes may depend on the lack of VEGF-C expression.

Arsenic speciation for the phytoremediation by the Chinese brake fern, Pteris vittata.

Arsenic (As) speciation for the phytoremediation by the Chinese brake fern was studied. In particular, the mechanism of how plants induce compounds containing thiol (SH) and proteins by As exposure in terms of the relationship between As and phosphate uptaken into plant cells was examined. Pteris vittata callus could efficiently reduce As(V) to As(III) by the rapid introduction of reductase and synthesize thiols leading to phytochelatins production. Furthermore, Pteris vittata could control phosphate concentration in the cells corresponding to the concentration of arsenite and arsenate. To our best knowledge, this is the first report to show the mechanisms of such high As tolerance of Pteris vittata using their callus in terms of in vitro approach for the analysis of As speciation and metabolism route.

Development of a new enzyme-linked immunosorbent assay for the detection of anti-M2 in primary biliary cirrhosis.

We developed a new enzyme-linked immunosorbent assay (ELISA) for the detection of antimitochondrial antibody (AMA)-M2 in sera from patients with primary biliary cirrhosis (PBC), using 2-oxo-acid dehydrogenase complex (2-OADC) purified from porcine myocardium as the antigen source. The immunoreactivity was tested in a total of 354 sera, including 63 sera from patients with PBC by our ELISA. In the sera, indirect immunofluorescence for AMA, former ELISA for anti-pyruvate dehydrogenase complex (PDC) and immunoblot assay were performed, respectively. Of the 63 sera from patients with PBC, 51 sera (81.0%) were positive for anti-M2 in the new ELISA. Thirty-eight of the 63 sera (60.3%) were positive for anti-PDC in the former ELISA; the difference was significant between them (P=0.011). None of the 291 control sera from healthy volunteers showed reactivity against 2-OADC in the new ELISA. Moreover, in comparison with the results of immunoblot analysis, sensitivity and specificity in our ELISA to the sera from patients with PBC were 100 and 92.3%, respectively. Our results indicate that the new ELISA for anti-M2 using 2-OADC is simple, rapid and sensitive enough for the detection of AMA specific to PBC.

False positive reaction in ELISA for IgM class anti-M2 antibody and its prevention.

Anti-M2 of anti-mitochondrial antibodies is recognized as the specific autoantibody detected in sera from patients with primary biliary cirrhosis (PBC). The IgG class and IgM class of this antibody can be separately measured using each ELISA. In the present study, false positive reactions were found in some sera from non-PBC patients such as acute hepatitis A, syphilis and rheumatoid arthritis using the IgM anti-M2 ELISA. They showed an increase of polyclonal IgM, and positivity for IgM anti-cardiolipin or rheumatoid factors, respectively. So, we developed a means to prevent these false positive reactions. First, dilutions of test sera at 1:1000-fold were carried out in addition to the original method at 1:100-fold. Secondly, some blocking reagents were added into the buffer system. By serum dilution, non-specific bindings disappeared in most samples other than showing an increase in polyclonal IgM. Moreover, the addition of suitable blocking reagents such as fetal bovine serum (FBS) and skimmed milk into the buffer system could prevent these non-specific bindings. From these findings, the procedure of optical serum dilution and the addition of suitable blocking reagents successfully prevented false positive reactions in this IgM anti-M2 ELISA.

High sensitivity of a novel ELISA for anti-M2 in primary biliary cirrhosis.

The use of an ELISA for the detection of anti-M2, a specific autoantibody in primary biliary cirrhosis (PBC), has been common in Japan. However, there are some problems in the sensitivity of this ELISA, especially in PBC patients showing antimitochondrial antibody (AMA)-negative sera or low AMA titers by immunofluorescence. Recently, a new ELISA for anti-M2 was developed, using porcine heart mitochondrial protein as the antigen. We report here comparative studies of the new and the former anti-M2 ELISAs. Porcine heart mitochondrial protein was prepared and used as the antigen for the new ELISA for anti-M2. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this protein showed three major M2 antigen proteins. As the second antibody, peroxidase-conjugated anti-human mouse monoclonal IgM, in addition to monoclonal IgG, was included. The sera of 171 PBC patients were examined. As controls, we examined the sera of 167 non-PBC patients and the sera of 115 normal controls. The cut-off index was set at 10 U/ml, based on the results for the normal controls. No sera from the non-PBC patients or the normal controls were positive for anti-M2 by either the new or the former ELISA. However, the positivity rate for anti-M2 in PBC patients with the new ELISA was 78%; in contrast, that with the former ELISA was only 54%; this difference was significant (P = 0.00001). In particular, in 65 patients showing AMA titers of 1:20 or less, the positivity rate with the new ELISA was 51%; in contrast, that with the former ELISA was only 17%. As the sensitivity of the new ELISA is significantly higher than that of the former ELISA, especially for sera from patients showing AMA-negativity or low titers of AMA, the new ELISA is considered to be more effective than the former ELISA for use in anti-M2 screening assays in patients with PBC.

A highly sensitive high-performance liquid chromatography-- fluorometric method for the assay of peptidylarginine deiminase activity.

The activity of peptidylarginine deiminase (PAD) has generally been assayed by a colorimetric method using N-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-arginine (Bz-L-Arg) as the substrates. The widespread occurrence of citrulline and urea in tissues makes use of this method difficult, especially for small samples. We developed a highly sensitive high-performance liquid chromatography method with N-dansyl-glycyl-L-arginine as the substrate. This method was sensitive enough to determine previously undetectable activity of PAD in HL-60 cells. Two types of PAD (HL-60 cell and brain PAD) could be distinguished by differential competition, using either BAEE or Bz-L-Arg as a preferential substrate in the assay. These data indicate that the present method is applicable to many tissues.

Axonal transports of Boc-Gly-Arg-Arg-MCA hydrolysing enzyme in rat sciatic nerves.

Study on neural axon transport is a very useful method to find a neuron-specific protease. In the present study, the enzyme activity (release of 7-amino-4-methyl-coumarin from t-butyloxycarbonyl-glycyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide) was measured in the proximal, middle, and distal segments between 12 and 120 h after double ligations of rat sciatic nerves to find precursor processing enzyme specific for pair of basic amino acid residue. The enzyme activity was significantly increased not only in the proximal but also in the distal segments 12-120 h after the ligation, and the maximal enzyme activity was found in both segments at 72 h. The enzyme activity eluted by anion exchange chromatography of the proximal segment showed at least three peaks, and was slightly higher than the activity of the distal one. The activity in the middle segment was very low in comparison with the activity in the proximal and distal segments. These data indicate that some of the enzymes specific for pair of basic amino acid residue are transported by both anterograde and retrograde axonal flow, and may undergo a neuron-specific processing.

The Japanese health care system: citizen complaints, citizen possibilities.

The Japanese health care system is sometimes considered one of the best in the world because it appears to have achieved universal coverage, high quality, and a comparatively low level of expenditure. But under compulsory national health insurance and the uniform fee schedule which has worked well so far, various problems have been produced in Japan. A growing number of persons believe some reform or readjustment may be required. Following a brief review of the Japanese health care system which includes health insurance mechanisms, the relationship among physicians, hospitals and clinics, and the impact of these structures on access to care are explored. The resulting cost of care and the quality of care are then addressed. The lack of consumer information and the nature of the physician-patient relationship related to cultural factors are important components of this health care system. These latter factors are in the process of change and the likely direction of their influence upon the Japanese health care system is explored.

[Development of novel clinical tests and their contribution to laboratory diagnosis--ligase chain reaction].

There are several methods that have been developed for the detection of a small amount of nucleic acids. Ligase chain reaction(LCR) is a highly sensitive assay for the detection of a specific DNA sequence. LCR utilizes four oligodeoxynucleotides(probes) complementary to each of target DNA strands and repeats a thermalcycling amplification step. Two pairs of oligodeoxynucleotides renature adjacent to one another on each of the separated target DNA strands, resulting in nick formation. A thermostable DNA ligase joins the nick covalently. Each ligated product can serve as a template sequence in subsequent rounds of thermalcycling (denaturation, annealing/ligation). In this manner LCR accumulates the ligated products exponentially by repeating thermalcycling. One of the distinctive features of LCR is to be able to detect a known point-mutation easily. Gap-LCR, a modified LCR, has been developed to improve the sensitivity and the specificity of the standard LCR by setting up gaps (3 < or = nucleotides) at the ligatable 3' and 5' termini of the probes. The gap-LCR probes form a short gap after annealing of the probes to the target DNA. The gap is filled in by a thermostable DNA polymerase and the resultant nick is sealed by a thermostable DNA ligase to generate the ligated probes. Gap-LCR repeats this step on thermalcycling and accumulates the LCR products in an exponential fashion.

High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity.

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Inhibin alpha, beta A and beta B subunit messenger ribonucleic acid levels in cultured rat pituitary: studies by a quantitative RT-PCR.

Recent studies have demonstrated that inhibins and activins both play not only endocrine roles but also local regulatory roles in gonadotoropin secretion. There has been controversy as to the subtype of rat pituitary inhibin/activin. We studied the levels of inhibin alpha, beta A and beta B subunit mRNAs by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) and the changes in their levels by adding inhibin alpha, beta A and beta B mRNA antisense oligonucleotides and inhibin A, activin A or GnRH to cultured rat anterior pituitary cells. This study demonstrated the level of 3 mRNAs to be 1.6 x 10(-2), 0.75 and 3.4 x 10(-2) molecules/cell with a molar ratio of 1:50:2. A stimulatory role for activin B in FSH secretion was suggested as beta B mRNA antisense oligonucleotide decreased FSH secretion. The beta B mRNA level tended to be decreased by the addition of activin A, but the decrease was not statistically significant. GnRH did not affect alpha and beta B mRNA levels when administered singly. The level of beta A mRNA was not changed by any of the above treatments. In conclusion, the presence of inhibin alpha, beta A and beta B subunit mRNAs in the rat anterior pituitary with the greatest abundance of beta A was demonstrated by using RT-PCR. Activin B or activin AB may play important roles in FSH secretion in an autocrine or a paracrine fashion, and activin A may play an indirect role in FSH secretion.

Demonstration of activin in normal pituitary and in various human pituitary adenomas by immunohistochemistry.

Inhibins and activins have been known to modify the secretion of various pituitary hormones. To study whether inhibins and activins are present in human pituitary tissues, immunohistochemical studies with antisera to activin A and inhibin alpha subunit were performed on 9 human pituitary adenoma tissue specimens and one sample of normal pituitary tissue adjacent to one adenoma. Activin immunoreactivities were demonstrated in the cytoplasms of one GH and one PRL and two non-functioning adenomas and one normal pituitary tissue, but they were negative in one PRL, one ACTH, one FSH and two non-functioning adenomas. Thus, the presence and absence of activin in the same type of adenoma in regard to hormone production, suggested that the difference in immunostaining simply reflected the difference in the activin concentration. In contrast to this, inhibin alpha subunit immunoreactivity was not found in any of the tissues studied. These data suggested a local synthesis of activin in the normal pituitary as well as various kinds of pituitary adenoma tissues and its local role in the human pituitary gland.

Inhibin alpha and beta A subunit messenger ribonucleic acids expression in human pituitary adenomas: studies by quantitative reverse transcription-polymerase chain reaction.

Inhibin alpha and beta A subunit messenger ribonucleic acid (mRNA) levels were measured quantitatively by reverse transcription-polymerase chain reaction (RT-PCR) in human pituitary adenomas. The inhibin alpha subunit mRNA levels were undetectably low in cultured adenoma tissues, but beta A mRNA were 0.383 +/- 0.074 in 3 GH adenomas, 0.672 +/- 0.140 in 3 prolactinomas and 0.957 +/- 0.414 molecules/cell in 3 non-functioning adenomas. The addition of 10(-8) M activin A decreased the beta A mRNA levels within 4 h in 1 of 3 GH adenomas, 2 of 3 prolactinomas and 2 of 3 non-functioning adenomas, though the decreases were not statistically significant. The results showed an abundance of beta A subunit mRNA compared with alpha subunit mRNA in all human pituitary adenomas and a local role for activin in its own production through inhibin beta A mRNA subunit expression.

A fluorometric assay for measuring deamidase (lysosomal protective protein) using high-performance liquid chromatography.

A rapid and sensitive assay method for the determination of deamidase activity is reported. This method is based on fluorometric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val (N-Dns-D-Tyr-Val), enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val-NH2 (N-Dns-D-Tyr-Val-NH2), after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure N-Dns-D-Tyr-Val at concentrations as low as 100 fmol, yields highly reproducible results and requires less than 8.5 min per sample for separation and quantitation. The optimum pH for deamidase activity was 4.0-4.5. Greater than 5 mM of reduced glutathione was needed for maximal enzyme activity. The Km and Vmax values were respectively 125 microM and 14.12 pmol/micrograms/h with the use of enzyme extract obtained from mouse spleen. Deamidase activity was strongly inhibited by Ag+, Cu2+, diisopropylfluorophosphate, and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in spleen. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this enzyme.

Immunological detection of penicillin-binding protein 2' of methicillin-resistant staphylococci by using monoclonal antibodies prepared from synthetic peptides.

Two synthetic peptides 31 and 32 amino acids in length were prepared as deduced from a known amino acid sequence of penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus. Two monoclonal antibodies were generated from fused cells of myeloma cells and splenic cells of mice immunized with the synthetic peptides. Western blot (immunoblot) analysis demonstrated specific binding of the antibodies to PBP2' of a methicillin-resistant S. aureus strain. An immunoradiometric assay was developed by using these antibodies for simple detection of PBP2'.

Inhibin alpha, beta A subunit and activin type II receptor mRNAs are expressed in human brain tumors.

Inhibin and activin were initially isolated as regulators of pituitary or gonadal hormone and are now known to be growth factors belonging to the TGF-beta family with diverse influences on the differentiation and proliferation of various tissues. To investigate the role of inhibin and activin in human brain tumors, the expression of inhibin alpha, and beta A mRNA as well as activin type II receptor (ACTR II) mRNA were studied in various human brain tumors. The tumors were divided into the following 4 groups: 3 Rathke's cleft cysts and 2 craniopharyngiomas (group 1), 8 meningiomas (group 2), 8 malignant gliomas (group 3), and various other tumors including 1 each of germinoma, astrocytoma, hemangioblastoma, and osteochondroma as well as 2 malignant lymphomas and 2 metastatic squamous cell carcinomas (group 4). Immediately after resection, tumor tissues were homogenized in guanidine thyiocyanate to extract total RNA. PCR was then performed with reverse-transcribed cDNA and the respective amplification primers. DNA bands were obtained by agarose gel electrophoresis. Messenger RNA for the inhibin beta A subunit was demonstrated in all of the tissues studied. In contrast, inhibin alpha subunit mRNA was expressed in 60%, 50%, 75%, and 75% of the tumors in groups 1, 2, 3 and 4, respectively, whereas ACTR II mRNA was demonstrated in 20%, 37.5%, 62.5% and 50% of the tumors in each group. Coexpression of mRNAs for the inhibin alpha, and beta A subunits and ACTR II occurred in some brain tumors. The levels of inhibin alpha and ACTR II mRNA tended to be higher in the tumors with a higher grade of malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly sensitive high-performance liquid chromatography-fluorimetric assay method for carboxypeptidase H activity.

A rapid and sensitive high-performance liquid chromatographic (HPLC)-fluorimetric assay method has been developed for the determination of carboxypeptidase H activity based on the measurement of N-(5-dimethyl-aminonaphthalene-1-sulfonyl)glycine (dansyl-Gly) formed enzymatically from dansyl-Gly-L-Lys or dansyl-Gly-L-Arg. Dansyl-Gly is eluted faster than the substrates with an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer at pH 7.0 containing methanol, but eluted slower with an acidic buffer at pH 4.6. The new HPLC method separates the product and substrate in less than 5 min using an elution buffer at pH 7.0 containing 60% methanol. Using this method carboxypeptidase H activity has been detected in rat sciatic nerves. This HPLC method facilitates the assay of carboxypeptidase H activity in the enzyme samples from various tissues.

Expression of inhibin alpha, and beta A subunit and activin type II receptor mRNAs in various human pituitary adenomas.

Inhibin and activin are known to be involved in the pituitary hormone secretion as well as proliferation of the pituitary. We studied the expression of inhibin alpha, and beta A subunit and activin type II receptor (ACTR 2) mRNAs in human pituitary adenomas to determine the significance of inhibin and activin in pituitary hormone secretion. Tumor tissues were homogenized immediately after resection in guanidinium thiocyanate to extract total RNA. PCR was performed with reversely transcripted cDNA and respective amplification primers. DNA bands obtained for inhibin alpha, beta A and ACTR 2 by agarose gel-electrophoresis were 367, 285, and 389 bp, respectively. Messenger RNAs for inhibin beta A were demonstrated in all of the pituitary tissues studied, namely in 3 GH, 2 ACTH, 6 PRL and 1 FSH producing adenomas and 17 non-functioning adenomas. Inhibin alpha mRNAs were detected in 10 of 12 functioning adenomas and 15 of 17 non-functioning adenomas. ACTR 2 mRNAs were found in 11 out of 17 non-functioning adenomas, but only found in 3 out of 12 functioning adenomas. These results suggested local production of activin, a homodimer of beta-subunits, and inhibin, a heterodimer of alpha and beta subunits, in most of the pituitary adenomas regardless of their hormone secretion. On the other hand, a significantly higher incidence of ACTR 2 in non-functioning adenomas than in functioning adenomas suggested that activin had its main site of action in non-functioning adenomas, which could be potential gonadotropinomas.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with antibodies against synthetic peptides derived from penicillin-binding protein 2'.

Ten kinds of peptides (21 to 32 amino acids in length) were synthesized based on the reported amino acid sequences of the penicillin-binding protein 2' (PBP2') of methicillin-resistant Staphylococcus aureus (MRSA). Antibodies against these synthetic peptides (SPs) were generated by immunizing rabbits. The antibodies raised against all the peptides except for one reacted to PBP2' of MRSA and to SPs used for immunization but not to any other protein of MRSA or methicillin-susceptible S. aureus (MSSA) tested by ELISA and Western blotting. A sandwich immunoradiometric assay (IRMA) for the detection of PBP2' was developed using these antibodies. The method could detect PBP2' extracted from as few as 3 x 10(4) cells of a clinical MRSA isolate, and a good correlation between cell number and signal radio-count was observed. IRMA was positive for all 51 methicillin-resistant staphylococci isolated from patients, and was negative for all the 28 methicillin-susceptible ones and 19 strains of other bacterial species. IRMA could be a simple and reliable method for MRSA detection in the clinical bacterial laboratory.

A rapid anterograde axonal transport of carboxypeptidase H in rat sciatic nerves.

Using the highly sensitive HPLC-fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative prohormone processing enzyme that removes a basic amino acid from the C-terminus of a precursor peptide, was measured 12-72 h after double ligations of rat sciatic nerves. CPH-like activity in rat sciatic nerves was 60-fold lower than that in the pituitary gland. CPH-like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is approximately 5 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.