Prognostic factors for survival in patients with pT1 N+ or T2-3 N0 gastric cancer in Japan.

BACKGROUND: The outcome for pT1 N+ or pT2-3 N0 gastric cancer is favourable, but some patients suffer from recurrent disease. The aim of this study was to identify prognostic factors in patients with pT1 N+ or pT2-3 N0 gastric cancer. METHODS: This was a multicentre, retrospective cohort study. All patients with pT1 N+ or pT2-3 N0 gastric cancer who underwent curative gastrectomy at five high-volume, specialized cancer centres in Japan between 2000 and 2008 were included. Demographic, clinical, surgical and pathological data were collected. Independent prognostic factors were identified using a Cox proportional hazards regression model. RESULTS: Some 1442 patients were included. The 5-year overall survival rate for patients with pT1 N+ or pT2-3 N0 gastric cancer was 92·0 per cent. Multivariable analysis for overall survival identified age (hazard ratio (HR) 2·67, 95 per cent c.i. 2·09 to 3·43), sex (HR 0·57, 0·39 to 0·83) and clinical tumour depth (cT) (HR 1·45, 1·06 to 1·98) as independent prognostic factors. CONCLUSION: Survival of patients with pT1 N+ or pT2-3 N0 gastric cancer is good. Age 65 years or above, male sex and cT2-4 category are associated with worse overall survival.

Significance of histologic pattern of carcinoma and sarcoma components on survival outcomes of uterine carcinosarcoma.

BACKGROUND: To examine the effect of the histology of carcinoma and sarcoma components on survival outcome of uterine carcinosarcoma. PATIENTS AND METHODS: A multicenter retrospective study was conducted to examine uterine carcinosarcoma cases that underwent primary surgical staging. Archived slides were examined and histologic patterns were grouped based on carcinoma (low-grade versus high-grade) and sarcoma (homologous versus heterologous) components, correlating to clinico-pathological demographics and outcomes. RESULTS: Among 1192 cases identified, 906 cases were evaluated for histologic patterns (carcinoma/sarcoma) with high-grade/homologous (40.8%) being the most common type followed by high-grade/heterologous (30.9%), low-grade/homologous (18.0%), and low-grade/heterologous (10.3%). On multivariate analysis, high-grade/heterologous (5-year rate, 34.0%, P = 0.024) and high-grade/homologous (45.8%, P = 0.017) but not low-grade/heterologous (50.6%, P = 0.089) were independently associated with decreased progression-free survival (PFS) compared with low-grade/homologous (60.3%). In addition, older age, residual disease at surgery, large tumor, sarcoma dominance, deep myometrial invasion, lymphovascular space invasion, and advanced-stage disease were independently associated with decreased PFS (all, P < 0.01). Both postoperative chemotherapy (5-year rates, 48.6% versus 39.0%, P < 0.001) and radiotherapy (50.1% versus 44.1%, P = 0.007) were significantly associated with improved PFS in univariate analysis. However, on multivariate analysis, only postoperative chemotherapy remained an independent predictor for improved PFS [hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.27-0.43, P < 0.001]. On univariate analysis, significant treatment benefits for PFS were seen with ifosfamide for low-grade carcinoma (82.0% versus 49.8%, P = 0.001), platinum for high-grade carcinoma (46.9% versus 32.4%, P = 0.034) and homologous sarcoma (53.1% versus 38.2%, P = 0.017), and anthracycline for heterologous sarcoma (66.2% versus 39.3%, P = 0.005). Conversely, platinum, taxane, and anthracycline for low-grade carcinoma, and anthracycline for homologous sarcoma had no effect on PFS compared with non-chemotherapy group (all, P > 0.05). On multivariate analysis, ifosfamide for low-grade/homologous (HR 0.21, 95% CI 0.07-0.63, P = 0.005), platinum for high-grade/homologous (HR 0.36, 95% CI 0.22-0.60, P < 0.001), and anthracycline for high-grade/heterologous (HR 0.30, 95% CI 0.14-0.62, P = 0.001) remained independent predictors for improved PFS. Analyses of 1096 metastatic sites showed that carcinoma components tended to spread lymphatically, while sarcoma components tended to spread loco-regionally (P < 0.001). CONCLUSION: Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.

Development and external validation of a nomogram for overall survival after curative resection in serosa-negative, locally advanced gastric cancer.

BACKGROUND: Few nomograms can predict overall survival (OS) after curative resection of advanced gastric cancer (AGC), and these nomograms were developed using data from only a few large centers over a long time period. The aim of this study was to develop and externally validate an elaborative nomogram that predicts 5-year OS after curative resection for serosa-negative, locally AGC using a large amount of data from multiple centers in Japan over a short time period (2001-2003). PATIENTS AND METHODS: Of 39 859 patients who underwent surgery for gastric cancer between 2001 and 2003 at multiple centers in Japan, we retrospectively analyzed 5196 patients with serosa-negative AGC who underwent Resection A according to the 13th Japanese Classification of Gastric Carcinoma. The data of 3085 patients who underwent surgery from 2001 to 2002 were used as a training set for the construction of a nomogram and Web software. The data of 2111 patients who underwent surgery in 2003 were used as an external validation set. RESULTS: Age at operation, gender, tumor size and location, macroscopic type, histological type, depth of invasion, number of positive and examined lymph nodes, and lymphovascular invasion, but not the extent of lymphadenectomy, were associated with OS. Discrimination of the developed nomogram was superior to that of the TNM classification (concordance indices of 0.68 versus 0.61; P < 0.001). Moreover, calibration was accurate. CONCLUSIONS: We have developed and externally validated an elaborative nomogram that predicts the 5-year OS of postoperative serosa-negative AGC. This nomogram would be helpful in the assessment of individual risks and in the consideration of additional therapy in clinical practice, and we have created freely available Web software to more easily and quickly predict OS and to draw a survival curve for these purposes.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Clinical significance of miR-144-ZFX axis in disseminated tumour cells in bone marrow in gastric cancer cases.

BACKGROUND: We previously reported that bone marrow (BM) was a homing site for gastric cancer (GC) cells leading to haematogenous metastases. There has been little study that microRNAs regulated pathways in malignant cells or host cells in BM, and thereby regulated the progression of GC. METHODS: Both microRNA microarray and gene expression microarray analyses of total RNA from BM were conducted, comparing five early and five advanced GC patients. We focused on miR-144-ZFX axis as a candidate BM regulator of GC progression and validated the origin of the microRNA expression in diverse cell fractions (EpCAM(+)CD45(-), EpCAM(-)CD45(+), and CD14(+)) by magnetic-activated cell sorting (MACS). RESULTS: Quantitative reverse-transcriptase (RT)-PCR analysis validated diminished miR-144 expression in stage IV GC patients with respect to stage I GC patients (t-test, P=0.02), with an inverse correlation to ZFX (ANOVA, P<0.01). Luciferase reporter assays in five GC cell lines indicated their direct binding and validated by western blotting. Pre-miR144 treatment and the resultant repression of ZFX in GC cell lines moderately upregulated their susceptibility to 5-fluorouracil chemotherapy. In MACS-purified BM fractions, the level of miR-144 expression was significantly diminished in disseminated tumour cell fraction (P=0.0005). Diminished miR-144 expression in 93 cases of primary GC indicated poor prognosis. CONCLUSION: We speculate that disseminated cancer cells could survive in BM when low expression of miR-144 permits upregulation of ZFX. The regulation of the miR-144-ZFX axis in cancer cells has a key role in the indicator of the progression of GC cases.

Long-term outcome after proximal gastrectomy with jejunal interposition for suspected early cancer in the upper third of the stomach.

BACKGROUND: Proximal gastrectomy was introduced as a function-preserving operation for early gastric cancer (EGC). The aim of this study was to investigate long-term outcomes after this procedure. METHODS: Between 1993 and 2005, patients with suspected EGC in the upper third of the stomach underwent proximal gastrectomy. The long-term oncological and surgical outcomes were assessed. RESULTS: Of 128 patients thought to have EGC, 14 had advanced disease. Nodal involvement was seen in 13 patients (10.2 per cent). Postoperative complications developed in 20 (15.6 per cent). Anastomotic stricture was the most frequent complication, occurring in 13 patients (10.2 per cent). There were no postoperative deaths. During follow-up, nine patients (7.0 per cent) were hospitalized owing to bowel obstruction. Eight (6.3 per cent) developed a second primary gastric carcinoma. The overall 5-year survival rate was 90.5 per cent. CONCLUSION: Proximal gastrectomy is well tolerated, with excellent outcomes in patients with suspected EGC. It is recommended as a standard procedure for the treatment of EGC in the upper third of the stomach.

Long-term results of gastrectomy for alpha-fetoprotein-producing gastric cancer.

BACKGROUND: alpha-Fetoprotein (AFP)-producing gastric cancer is a rare tumour. It is said to have a high incidence of liver metastasis and poor prognosis. This study sought to evaluate long-term outcomes in such patients. METHODS: Records of consecutive patients with gastric carcinoma who underwent preoperative measurement of serum AFP levels and gastrectomy were reviewed to identify those who satisfied the following criteria: preoperative AFP level exceeding 40 ng/ml with a decrease after gastrectomy, or raised preoperative AFP level (10-39 ng/ml) and resected tumour showing histologically characteristic features or immunohistochemically positive AFP production. RESULTS: Of 3374 patients with gastric cancer, 53 (1.6 per cent) met the selection criteria. Tumours were characterized by a high incidence of nodal (79 per cent) or liver (53 per cent) metastasis. Preoperative serum AFP levels showed no correlation with tumour size, depth of invasion, disease stage or survival. The 5-year survival rate was 34 per cent. Five patients survived after recurrence following multimodal treatment. A rising AFP level during follow-up always led to tumour recurrence, but the level remained normal in 11 of 31 patients with recurrence. CONCLUSION: AFP-producing tumours represent a small subgroup of gastric cancer with high metastatic potential. Postoperative serum AFP level can help predict recurrence but a normal level does not mean absence of recurrence. Prognosis is not as poor as previously thought, and multimodal treatment may be worthwhile even in patients with recurrent tumour.

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Expression of uPAR mRNA in peripheral blood is a favourite marker for metastasis in gastric cancer cases.

Urokinase-type plasminogen activator receptor (uPAR) plays a central role in the plasminogen activation cascade and participates in extracellular matrix degradation, cell migration and invasion. We evaluated the expression level of uPAR mRNA and the presence of isolated tumour cells (ITCs) in bone marrow (BM) and peripheral blood (PB) in gastric cancer patients and clarified its clinical significance. We assessed specific uPAR mRNA expression by quantitative real-time reverse transcriptase- polymerase chain reaction (RT-PCR) in BM and PB in 846 gastric cancer patients as well as three epithelial cell markers, carcinoembryonic antigen (CEA), cytokeratin (CK)-19 and CK-7. The uPAR mRNA expression in bone marrow and peripheral blood expressed significantly higher than normal controls (P<0.0001). The uPAR mRNA in BM showed concordant expression with the depth of tumour invasion, distant metastasis, and the postoperative recurrence (P=0.015, 0.044 and 0.010, respectively); whereas in PB, we observed more intimate significant association between uPAR expression and clinicopathologic variables, such as depth of tumour invasion, the distant metastasis, the venous invasion and the clinical stage (P=0.009, 0.002, 0.039 and 0.008, respectively). In addition, the uPAR mRNA expression in PB was an independent prognostic factor for distant metastasis by multivariate analysis. We disclosed that it was possible to identify high-risk patients for distant metastasis by measuring uPAR mRNA especially in peripheral blood at the timing of operation in gastric cancer patients.

Bone marrow and peripheral blood expression of ID1 in human gastric carcinoma patients is a bona fide indicator of lymph node and peritoneal metastasis.

Recent studies have showed that the bone marrow-derived endothelial progenitor cells play critical roles in metastasis and that ID1 is required in metastasis as regulator of angiogenesis. Therefore, we investigated the clinical significance of ID1 mRNA expression in bone marrow and peripheral samples in patients with gastric cancer. Two hundred and eighty-nine bone marrow and 196 peripheral blood samples from gastric cancer patients were collected and analysed by quantitative RT-PCR for ID1. The ID1 protein expression in one bone marrow, three metastatic lymph nodes and three peritoneal disseminated tumours was examined by immunohistochemical methods. In both bone marrow and peripheral blood samples, ID1 mRNA expression in the metastatic group was significantly higher than in any other group (P=0.003, P=0.0001, respectively) and significantly associated with lymph node metastasis and peritoneal dissemination. The cells in bone marrow with metastatic cancer stained strongly with ID1 compared with those of healthy volunteers. The expression of ID1 mRNA in bone marrow and peripheral blood was significantly associated with lymph node metastasis and peritoneal dissemination, and therefore constitutes a predictable marker for lymph node metastasis and peritoneal dissemination.

The prognostic impact of isolated tumor cells in lymph nodes of T2N0 gastric cancer: comparison of American and Japanese gastric cancer patients.

BACKGROUND: The clinical significance of immunohistochemically detected isolated tumor cells (ITC) in lymph nodes of gastric cancer patients is controversial. This study examined the prognostic impact of ITC on patients with early-stage gastric cancer in two large volume centers in the United States and Japan. METHODS: Fifty-seven patients with T2N0M0 gastric carcinoma who underwent gastric resection between January 1987 and January 1997 at Memorial Sloan-Kettering Cancer Center (MSKCC) in New York and 107 patients resected at National Cancer Center Hospital (NCCH) in Tokyo between January 1984 and December 1990 were studied. The sections were newly prepared from each lymph node for immunohistochemical staining for cytokeratin. Lymph nodes and original specimens from MSKCC were examined by pathologists in NCCH. The prognostic significance of the presence of ITC in lymph nodes was investigated in patients of both institutions. RESULTS: ITC were identified in 30 of 57 patients (52.6%) at MSKCC and in 38 of 107 patients (35.5%) at NCCH. In both institutions, there was no significant difference in the prognosis of the studied patients with or without ITC (P= .22, .86 respectively). CONCLUSIONS: The presence of ITC detected by immunohistochemistry in the regional lymph nodes did not affect the prognosis of American and Japanese patients with T2N0M0 gastric carcinoma who underwent gastrectomy with D2 lymph node dissection.

Outcome of pylorus-preserving gastrectomy for early gastric cancer.

BACKGROUND: Pylorus-preserving gastrectomy has been introduced as a function-preserving operation for early gastric cancer in Japan. The aim of this study was to investigate the safety and radicality of the procedure. METHODS: Between 1995 and 2004, 611 patients with apparent early gastric cancer in the middle third of the stomach had pylorus-preserving gastrectomy. The short-term surgical and long-term oncological outcomes of these operations were assessed. RESULTS: The accuracy of preoperative diagnosis of early gastric cancer was 94.3 per cent. Nodal involvement was seen in 62 patients (10.1 per cent). There were no postoperative deaths. Complications developed in 102 patients (16.7 per cent). Major complications, such as leakage and abscess, were observed in 19 (3.1 per cent). The most common complication was gastric stasis, occurring in 49 (8.0 per cent). The overall 5-year survival rate in patients with early gastric cancer was 96.3 per cent. CONCLUSION: Pylorus-preserving gastrectomy is a safe operation with an excellent prognosis in patients with early gastric cancer. It is recommended as the standard procedure for early gastric cancer in the middle third of the stomach.

Treatment strategy after non-curative endoscopic resection of early gastric cancer.

BACKGROUND: Endoscopic resection (ER) is indicated for patients with early gastric cancer who have a negligible risk of lymph node metastasis (LNM). Histological examination of the resected specimen may indicate a possible risk of LNM or a positive resection margin. These patients are considered to have undergone non-curative ER. The aim of this study was to determine the appropriate treatment strategy for such patients. METHODS: A total of 298 patients who had non-curative ER were classified into those with a positive lateral margin only (group 1; 72 patients) and those with a possible risk of LNM (group 2; 226 patients). RESULTS: Surgery was performed within 6 months of non-curative ER in 19 patients in group 1 and 144 in group 2. In group 1, nine patients were found to have local residual tumours, all limited to the mucosal layer without LNM. In Group 2, 13 patients had residual disease, including four local tumours without LNM, two local tumours with LNM and seven cases of LNM alone. The rate of LNM after surgery was 6.3 per cent in group 2. CONCLUSION: Surgery remains the standard treatment after non-curative ER in patients with a possible risk of LNM.

Precise switching of DNA replication timing in the GC content transition area in the human major histocompatibility complex.

The human genome is composed of long-range G+C% (GC%) mosaic structures thought to be related to chromosome bands. We previously reported a boundary of megabase-sized GC% mosaic domains at the junction area between major histocompatibility complex (MHC) classes II and III, proposing it as a possible chromosome band boundary. DNA replication timing during the S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify to the nucleotide sequence level the replication switch point during the S phase, we determined the precise DNA replication timing for MHC classes II and III, focusing on the junction area. To do this, we used PCR-based quantitation of nascent DNA obtained from synchronized human myeloid leukemia HL60 cells. The replication timing changed precisely in the boundary region with a 2-h difference between the two sides, supporting the prediction that this region may be a chromosome band boundary. We supposed that replication fork movement terminates (pauses) or significantly slows in the switch region, which contains dense Alu clusters; polypurine/polypyrimidine tracts; di-, tri-, or tetranucleotide repeats; and medium-reiteration-frequency sequences. Because the nascent DNA in the switch region was recovered at low efficiency, we investigated whether this region is associated with the nuclear scaffold and found three scaffold-associated regions in and around the switch region.

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells.

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G(1) phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.

The clinical significance of para-aortic nodal dissection for advanced gastric cancer.

BACKGROUND: Advanced gastric cancer with the risk of extensive nodal involvement has a poor prognosis even after radical surgery. We aimed to comprehensively review the clinical significance of extended radical dissection. METHODS: Between 1990 and 1999, 232 patients underwent radical gastrectomy with D2 plus para-aortic lymph node dissection at the National Cancer Center Hospital in Tokyo. We analyzed the short-term surgical and long-term oncological outcomes of these operations. RESULTS: Major complications occurred in 34 patients (14.7%). Median operation time was 325 min (range: 182-555) and median blood loss was 715 ml (range: 95-4457). There were 2 (0.9%) hospital deaths. Nodal involvement of the para-aortic area was seen in 33 patients (14.2%). The overall 5- and 10-year survival rates in patients with para-aortic node involvement were 21.2 and 15.2%, respectively. Multivariate analysis of patients with para-aortic node involvement revealed advanced age and metastasis in the interaorticocaval lymph nodes above the left renal vein to be independent risk factors impacting overall survival. CONCLUSIONS: PAN dissection has limited applicability and effectiveness to the patients with advanced gastric cancer. Above all, advanced age and metastasis in the interaorticocaval lymph nodes above the left renal vein are significant poor prognostic factor even after radical resection.

Gene structure, chromosomal localization and immunolocalization of chicken centromere proteins CENP-C and ZW10.

We determined the genomic structures and complete sequences of the coding regions of the chicken CENP-C and ZW10 genes. These two genes encode proteins that are thought to be involved in maintaining the fidelity of chromosome segregation. The chicken CENP-C gene is 30 kb in length and contains 19 exons. The chicken ZW10 gene spans 10 kb and contains 15 exons. The 5'-untranslated regions of these genes contain several binding sites for transcription factors such as Sp-1, E2F, p300, and members of the GATA family. By fluorescence in situ hybridization (FISH) analysis, the CENP-C was mapped to chromosome 4 and the ZW10 gene was mapped to a microchromosome. Antibodies against the chicken ZW10 protein revealed a cell cycle-dependent staining pattern in DT40 cells. ZW10 protein was distributed throughout the cytoplasm of DT40 cells during interphase. In most metaphase cells, ZW10 proteins appeared equally divided between the centromere and the spindle apparatus. During anaphase, chicken ZW10 proteins were no longer localized near chromosomes or the mitotic apparatus but were present diffusely in the cytoplasm.

Gene organization of human NOTCH4 and (CTG)n polymorphism in this human counterpart gene of mouse proto-oncogene Int3.

The cDNA and genomic clones for the human counterpart of the mouse mammary tumor gene Int3 were isolated and sequenced. We designated this human major histocompatibility complex (MHC) class III gene as NOTCH4, since very recently, by sequencing cDNA clones, the complete form of the mouse proto-oncogene Int3 has been clarified and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and by comparing it with the mouse Notch4 cDNA sequence, the exon/intron organization was clarified. The comparison of the predicted amino acid sequence of human NOTCH4 with those of other Notch homologues of a wide range of species revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)n repeats showing a variable number tandem repeat (VNTR) polymorphism for different human leukocyte antigen (HLA) haplotypes. Ten genes mapped on 6p21.3, including NOTCH4, were found to have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution. Similarity of genes on chromosomes 1, 6, 9 and 19 was also discussed.

Isolation and characterization of tributyltin chloride-resistant marine Vibrio.

Tributyltin chloride (TBTCl)-resistant marine bacteria were isolated from coastal sea water. One of these bacteria (Vibrio M-1) was highly resistant when grown in medium containing 125 microM of TBTCl. This strain was sensitive to other metals. Two polypeptides, 30 kDa and 12 kDa, increased when the strain was cultured in the medium supplemented with TBTCl. Initially TBTCl was taken up by the cell; however, the amount of TBTCl determined in the cellular fraction was low after the exponential growth phase of Vibrio M-1, suggesting the existence of a TBTCl-efflux system.

Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom.

In addition to phospholipase A2-I (PLA2-I) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA2-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+. Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II).