Phase I/II study of intraperitoneal docetaxel plus S-1 for the gastric cancer patients with peritoneal carcinomatosis.

PURPOSE: We designed a phase I/II trial of intraperitoneal (IP) docetaxel plus S-1 to determine the maximum tolerated dose (MTD) and recommended dose (RD) and to evaluate its efficacy and safety in gastric cancer patients with peritoneal carcinomatosis (PC). METHODS: Patients with PC confirmed by laparoscopy or laparotomy received IP docetaxel on days 1 and 15 and S-1 (80 mg/m(2)) on days 1-14 every 4 weeks. RESULTS: In the phase I part (n = 12), each cohort received escalating doses of docetaxel (35-50 mg/m(2)); the MTD was determined to be 50 mg/m(2) and the RD was determined to be 45 mg/m(2). Dose-limiting toxicities included grade 3 febrile neutropenia and grade 3 diarrhea. In the phase II part (n = 27), the median number of courses was 4 (range 2-11). The 1-year overall survival (OS) rate was 70 % (95 % confidence interval 53-87 %). The overall response rate was 22 % and peritoneal cytology turned negative in 18 of 22 (81 %) patients. The most frequent grade 3/4 toxicities included anorexia (19 %), neutropenia (7 %), and leukopenia (7 %). CONCLUSION: IP docetaxel plus S-1 is active and safety in gastric cancer patients with PC.

Anomalous union of the pancreaticobiliary ductal system without dilation of the common bile duct or tumor: case reports and literature review.

Among 698 patients subjected to endoscopic retrograde cholangiopancreatography, an anomalous union of the pancreaticobiliary ductal system (AUPBD) was found in 6 patients (0.9%). One of these 6 patients had an associated congenital choledochal cyst, and 4 did not. Two of these 4 patients, however, had advanced gallbladder cancer. The remaining 2 patients had no associated carcinoma of the biliary tract. A clinicopathological study was performed on these 2 patients and 26 such cases have been reported in the literature. The diagnosis of AUPBD was made by direct cholangiography. Regarding the type of union, the Pancreatic-Biliary type was present in 15 of 18 (83.3%) of these patients. Fifteen of 28 patients (53.6%) had right upper abdominal pain. Thickness of the gallbladder wall was visualized in 26 of the 28 (92.9%) patients. Gallstones were present in 4 (14.3%). AUPBD should always be kept in mind when a patient with abdominal pain is diagnosed as having gallbladder wall thickness without gallstones. ERCP should be performed in these patients in order to detect AUPBD without dilation of the bile duct. This may allow early detection of carcinoma of the biliary tract.

The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Amplification of the c-met, c-erbB-2 and epidermal growth factor receptor gene in human gastric cancers: correlation to clinical features.

We examined amplification of the c-met, c-erbB-2, and epidermal growth factor receptor (EGFR) gene in the patients with primary gastric cancer, and compared the data with clinical features in order to clarify the relationship between oncogenic abnormality and clinical features. Oncogene amplifications were examined by slot blot hybridization using DNAs extracted from formalin-fixed and paraffin-embedded tissues of primary gastric cancers. Seven of the seventy cancers (10.0%) had c-met gene amplification, nine (12.9%) had c-erbB-2 gene amplification, and six (8.6%) had EGFR gene amplification, respectively. Eighteen cases (25.7%) exhibited one or multiple oncogene amplification, and two cases (2.9%) exhibited simultaneous amplification of the three genes. The cases with c-met gene amplification tend to show invasive character and were related to peritoneal dissemination. The cases with c-erbB-2 gene amplification were related to lymph node metastasis. The cases with EGFR gene amplification had large tumors and were in highly advanced stage. The survival rate in patients with oncogene amplification was significantly lower than that in patients without amplification. Our data indicated that these genes were related to growth and metastasis of gastric cancer. Furthermore, this study about the three genes suggested that the type of activated gene might decide on the type of metastasis and clinical features.

The relation between the growth patterns of gastric carcinoma and the expression of hepatocyte growth factor receptor (c-met), autocrine motility factor receptor, and urokinase-type plasminogen activator receptor.

BACKGROUND: Hepatocyte growth factor receptor (c-met), autocrine motility factor receptor (AMFR), and urokinase-type plasminogen activator receptor (uPAR) are known to play important roles in tumor cell migration, invasion, and metastasis. The authors studied the relation between the expression patterns of these genes and the growth patterns of human gastric carcinoma. METHODS: The relation between the expression of c-met, AMFR, and uPAR and clinicopathologic parameters was studied using immunohistochemical preparations from 102 paraffin embedded primary gastric carcinomas. RESULTS: Of 102 cases, 43 (42%) had overexpression of c-met, and AMFR and uPAR immunoreactivity was observed in 41 cases (40%) and 38 cases (37%), respectively. Macroscopic examination revealed that all three genes were expressed in 1 (3%) of 32 early stage gastric carcinomas, 0 (0%) of 29 localized carcinomas (Borrmann types 1 and 2), and 16 (39%) of 41 infiltrating carcinomas (Borrmann types 3 and 4). In particular, the incidence (68%, 13 of 19 cases) of simultaneous expression of the three genes was significantly higher in Borrmann type 4 gastric carcinoma than in the other macroscopic types (P < 0.01). The overexpression of these genes was also closely associated with lymph node metastasis and peritoneal dissemination. In addition, the simultaneous overexpression of the three genes was associated with positive lymphatic vessel invasion and infiltrating type. Patients with tumors that simultaneously expressed all three genes had significantly poorer prognoses than those with tumors expressing only one or two of the genes. Furthermore, the number of genes expressed was closely related to the prognosis, and the Cox proportional hazards model identified this as one of the independent prognostic factors. CONCLUSIONS: These results suggest that the expression patterns of c-met, AMFR, and uPAR may be closely associated with the progression and invasion of gastric carcinoma as well as the prognoses of the patients. Borrmann type 4 gastric carcinoma is characterized by the diverse and simultaneous expression of these three genes.

Double cystic duct found by intraoperative cholangiography in laparoscopic cholecystectomy.

We report a rare case of double cystic duct in a 74-year-old woman. The patient complained of mild epigastric discomfort, and several stones were discovered by ultrasonography and computed tomography. Any anomalies of the biliary tract were undetectable in the preoperative examinations without direct cholangiography. Laparoscopic cholecystectomy was performed. After clipping the cystic duct close to the gallbladder, as usual, serial intraoperative cholangiography was performed and unexpectedly showed the inflow of contrast medium into the gallbladder via another cystic duct arising from the right hepatic duct, thus revealing one gallbladder and two cystic ducts, one of which joined the common hepatic duct and the other the right hepatic duct. There was only one cystic artery that arose from the right hepatic artery and accompanied the primary cystic duct to be distributed to the gallbladder. The existence of contrast medium in the resected specimen was confirmed by radiography. No complications occurred during or after laparoscopic cholecystectomy. This is the first report of double cystic duct found in laparoscopic cholecystectomy. We recommend routine preoperative or intraoperative cholangiography.

Expression of autocrine motility factor receptor correlates with disease progression in human gastric cancer.

Up-regulation of autocrine motility factor receptor (AMF-R) expression has been shown to be associated with invasion and metastasis of experimental tumour systems and human neoplasms. Monoclonal antibodies against AMF-R (gp78) were used to stain 221 primary gastric cancer specimens, and level of expression was examined in relation to pathological stage and prognostic values. In 125 out of 221 (56.6%) patients, gp78 was detected. Expression of gp78 was associated with macroscopic type, lymphatic and venous invasions, and lymph node and peritoneal metastasis. The level of gp78 expression in the cancer specimens was associated with histopathological stage and grade of tumour penetration. Positive gp78 expression was significantly associated with poor prognosis (P < 0.001). This significant relationship remained among patients in stage II and III. The results suggest that gp78 expression could be used as a prognostic marker in gastric cancer patients.

[Chemohyperthermic peritoneal perfusion and high-dose chemotherapy followed by peripheral blood stem cell transplantation in advanced colon cancer--a case report].

A 49-year-old woman who suffered from caecal cancer in 1988 underwent chemohyperthermic peritoneal perfusion for peritoneal and ovarian metastases in 1990, and high dose chemotherapy (HDC) with peripheral blood stem cell transplantation (PBSCT) for lung metastases in 1995. Heated saline containing anticancer drugs such as cisplatin, mitomycin C, etoposide (ETP), and pirarubicin, was intraperitoneally perfused at 43 degrees C for 60 minutes. The CD34 positive cells were mobilized by intravenous 500 micrograms G-CSF administration on five consecutive days. These cells were transplanted three days after the last day in the course of HDC, which included intravenous administration of 475 mg carboplatin, 2,020 mg cyclophosphamide, and 540 mg etoposide. The patient has survived with no sign of the disease.

Correlation between overexpression of c-met gene and the progression of gastric cancer.

The c-met oncogene encodes a receptor of hepatocyte growth factor (HGF), which has both mitogenic and motogenic activities. We have studied the c-met gene expression in gastric cancer by immunohistochemical method using an antibody specific to the c-met beta-chain. c-met immunoreactivity was preferentially localize on the cell membrane, and 55 (43%) of 127 primary gastric cancers showed c-met immunoreactivity. Strong correlation was found between c-met expression in large tumors, lymph node involvement, serosal invasion, as well as peritoneal dissemination. Furthermore, c-met positive tumors had a tendency to metastasize to more distant lymph node sites. Regarding the macroscopic type, Borrmann type 4 gastric cancer showed the highest incidence (12/16, 76%) of positive c-met expression. Patients with c-met positive tumor ran a significantly poorer prognosis than those with c-met negative one. These results indicate that the c-met protein participates in the progression and invasion of stomach cancer, and that c-met tissue status is a useful biological marker in gastric cancer.

Decreased expression of p16 gene in gastric cancer.

We analyzed gastric cancers for p16 gene alterations. Homozygous deletion of the p16 gene was found in 1 of 8 gastric cancer cell lines and none of 20 primary gastric cancers. Mutations of the p16 gene were not detected in exon 1-3. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed decreased or no expression of the p16 gene in 1 of 8 cell lines (12.5%) and in 8 of 20 tumors (40%), although no homozygous deletions or mutations within exon 1-3 were detected. These results suggest that the p16 gene may correlate to tumorigenesis due to decrease or loss of gene products in gastric cancers.

Cytoreductive surgery and sandwich therapy with chemohyperthermic peritoneal perfusion and intra-aortic chemotherapy for peritoneal dissemination in gastric cancer.

Cytoreductive resection (RST), chemohyperthermic peritoneal perfusion (CHPP) and/or intra-aortic chemotherapy (IA-chemo) were performed for peritoneal dissemination in gastric cancer. Ninety-six patients with peritoneal dissemination were grouped into tubercular (TB), 40; nodular (ND), 31; diffuse (DF) type, 19; and others, 6, respectively, by the gross findings. Sixty-three patients underwent RST. Fifty-nine patients received CHPP by 10-liter heated saline. Thirty patients underwent intra-aortic catheterization for the IA-chemo. The 1-year and 2-year survival rate (1-ysr and 2-ysr) of the RST(+) group were 47% and 10% significantly greater than the 9% and 0% of the RST(-) group (p<0.001). The 1-ysr and 2-ysr of the CHPP(+) group were 37% and 11% significantly greater than the 27% and 0% of the CHPP(-) group (p=0.04). In the TB type the 1-ysr and 2-ysr of the former was 43% and 8% significantly greater than the 15% and 0% of the latter (p=0.04). But there was no significant difference in survival time between the CHPP(+) and the CHPP(-) group in the ND type (p=0.22) or in the DF type (p=0.42). The 1-ysr and 2-ysr of the IA-chemo(+) group were 49% and 19% significantly greater than the 27% and 2% of the IA-chemo(-) group (p<0.01). In the DF type the 1-ysr and 2-ysr of the former was 50% and 33% significantly greater than the 8% and 0% of the latter (p=0.02). However, there was no significant difference in survival time between the IA-chemo(+) and the IA-chemo(-) group in the TB type (p=0.06) or in the ND type (p=0.50). Moreover, the effect of the combination therapy of CHPP and IA-chemo (the sandwich therapy, SDW) were examined. The 1-ysr and 2-ysr of the SDW(+) group were 49% and 22% significantly greater than the 24% and 0% of the SDW(-) group (p=0.002). The sandwich therapy should be performed in addition to cytoreductive surgery for improvement of prognosis in the patient with intractable peritoneal dissemination.

[Chemohyperthermic peritoneal perfusion and subtotal peritonectomy for peritonitis carcinomatosa in gastroenteric cancer].

A new operative procedure, called subtotal peritonectomy (SP), in combination with chemohyperthermic peritoneal perfusion, was developed for the treatment of peritonitis carcinomatosa in gastrointestinal cancer. SP includes resection of primary lesion, colon, small bowel, spleen, and gall bladder and parietal peritonectomy. Six patients with gastric cancer and two patients with colon cancer underwent these procedures. A great deal of discharge from the peritoneal cavity, an increase in systemic vascular resistance index, and a decrease in central venous pressure represented much decrease in circulatory volume on days 1 to 2 postoperatively. This state improved at 3 to 4 days after operation. Histopathological study revealed multiple peritoneal seedings with negative surgical margins in all patients. There were no related deaths though bleeding, perforation, and abscess occurred in two patients each. One patient died of peritoneal recurrence after one year, but the other have survived.

Amplification of epidermal growth factor receptor gene and its relationship to survival in human gastric cancer.

The correlation between the clinical features in 103 patients with primary gastric carcinoma and amplification of epidermal growth factor receptor (EGFR) gene was analyzed retrospectively. EGFR gene amplification was examined by slot-blot hybridization using DNA extracted from formalin-fixed, paraffin-embedded tissues. EGFR expression was also examined immunohistochemically using the same tissues with a monoclonal antibody that is monospecific for EGFR. In 5 of 103 cases (4.9%), a 2- to 11-fold amplification of EGFR gene was detected. Four of these 5 cases were poorly differentiated adenocarcinomas. All of them had overexpressions of EGFR. The cumulative survival rate of patients with EGFR gene amplification was significantly lower than that of the patients without amplification (p < 0.05) and all of them died within 3 years. Except for tumor size (p < 0.03), there were no significant clinicopathologic differences between the two groups. On the other hand, 41 of 103 cases (39.8%) exhibited expression of EGFR. However, there was no significant correlation between EGFR expression and clinicopathologic factors or prognosis. These results indicate that EGFR gene amplification may occur in advanced stages during the progression and be an important indicator of poor short-term prognosis in gastric carcinoma.

Prophylaxis with intraoperative chemohyperthermia against peritoneal recurrence of serosal invasion-positive gastric cancer.

Continuous hyperthermic peritoneal perfusion (CHPP) with a solution which contains 30 mg mitomycin C and 300 mg cisplatin has been introduced as a prophylactic treatment for peritoneal recurrence after curative resection of 79 advanced gastric cancers. The control group consisted of 81 patients with advanced gastric cancer who underwent curative surgery during the same period. CHPP was performed for 60 minutes by perfusing MMC- and CDDP-containing saline solutions warmed at 43.5 degrees C by a special CHPP device. In patients with pathologically confirmed serosal invasion-positive tumors, the survival rate of the CHPP group was significantly higher than that of the control group. A survival advantage for stage IV patients was also obtained by CHPP. However, there was no survival advantage between the CHPP group and the control group with serosal invasion-negative tumors. Adverse effects were observed in four patients who underwent CHPP: One developed severe bone marrow suppression, and transient hyperazotemia was observed in the other three. There was no difference in the incidence of mortality and morbidity between the two groups. These results indicate that CHPP is a safe, readily available prophylactic therapy for peritoneal recurrence after gastric cancer surgery.

Continuous hyperthermic peritoneal perfusion for the prevention of peritoneal recurrence of gastric cancer: randomized controlled study.

We performed continuous hyperthermic peritoneal perfusion (CHPP) or continuous normothermic peritoneal perfusion (CNPP) combined with cisplatin (CDDP) 300 mg/kg and mitomycin C (MMC) 30 mg/kg in an attempt to prevent peritoneal recurrence after surgery for gastric cancer. Twenty-two patients were treated with perfusion using about 10 liters of saline heated to 41 degrees to 42 degrees C (CNPP group); 18 patients were treated with saline heated to 37 degrees to 38 degrees C (CNPP group); and 18 patients underwent only gastric surgery without perfusion (control group) in a randomized control study. There were two deaths (9%) due to peritoneal recurrence in the CHPP group, four (22%) in the CNPP group, and four (22%) in the control group. The 1-, 2-, and 3-year survival rates were 95%, 89%, and 68%, in the CHPP group; 81%, 75%, and 51%, in the CNPP group; and 43%, 23%, and 23%, in the control group, respectively. There was a significant difference between the three survival curves by the log-rank test (p < 0.01). This difference showed that CNPP and CHPP are both effective procedures for preventing peritoneal recurrence. The maximum concentrations in the perfusate of total and free CDDP with 300 mg administration were 12.2 and 10.1 micrograms/ml, respectively, at the end of the perfusion, and the maximum concentrations of total and free CDDP in plasma were 2.1 and 1.0 micrograms/ml, respectively. The maximum concentrations of MMC in perfusate and plasma with 30 mg administration were 1.00 and 0.05 micrograms/ml, respectively, which are intraperitoneally cytotoxic but systemically safe concentrations.

[Correlation of DNA ploidy, c-erbB-2 protein tissue status, level of PCNA expression and clinical outcome in gastric carcinomas].

One-hundred and sixty-four patients with gastric carcinomas, who underwent gastrectomy during 1979-1985, were studied. Sixty-five of these cases were early gastric carcinomas, and the others were advanced gastric carcinomas, and the others were advanced gastric carcinomas. The nuclear DNA contents were measured by cytofluorometry, and immunohistochemical study on the expression of c-erbB-2 protein was performed using a monoclonal antibody against the c-erbB-2 oncogene product. Furthermore, immunohistochemical detection of proliferating cell nuclear antigen (PCNA) was performed using a monoclonal antibody against the PCNA. The rates of positive invasion beyond submucosal layer, lymphatic invasion, and vascular invasion in aneuploid cases were significantly higher than those in diploid ones, and the patients with aneuploid tumor had a significantly worse prognosis than those with diploid tumor. The rates of positive lymph node metastases and invasion beyond submucosal layer in the group with positive staining of the c-erbB-2 protein was significantly higher than in the negative group, and the group with positive staining for c-erbB-2 had a significantly worse prognosis than the negative one. PCNA indices showed a significant correlation with lymph node metastasis, and the group with higher PCNA indices had a worse prognosis. The patients with tumor showing both aneuploid and positive staining for c-erbB-2 protein, had the worst prognosis. There is a relationship between c-erbB-2 tissue status and PCNA indices, but no correlations were found among c-erbB-2 tissue status, PCNA indices and DNA contents. From these results, it can be concluded that DNA ploidy, c-erbB-2 protein, and PCNA may reflect the malignant potential of gastric carcinoma.

Correlation between altered expression of retinoblastoma protein and clinical outcome in patients with gastric-cancer.

We studied the expression of RB protein in 136 endoscopically obtained biopsied materials from primary gastric cancers by means of immunohistochemistry. The expression of RB protein was variably observed in the nuclei of tumor cells. Tumors could be divided into two groups. Group 1 (RB-positive) comprised 74 tumors (54%) that expressed RB protein in 20% or more of tumor cells, and Group 2 (altered RB) included 62 tumors (46%) that showed RB immunoreactivity in less than 20% of tumor cells. RB-positive tumors (Group 1) were more frequently observed in the early cancers (74%, 28/38) than in advanced ones (48%, 46/98). The incidence of distant metastases was higher and involvement of serosa or lymph nodes was more frequently observed in Group 2 than in Group 1. In addition, patients of Group 1 survived significantly longer than those of Group 2 (p<0.05). However, no correlation could be found between RB tissue status and proliferative activity. These results indicate that tumors with decreased RB-protein expression may have more aggressive biological behavior than tumors showing high expression of this protein.

Evidence of autocrine mechanism in poorly differentiated adenocarcinoma of the stomach.

To evaluate a potential role of an autocrine mechanism in gastric cancer, we carried out an immunohistochemical study of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), and epidermal growth factor receptor (EGFR) in 167 primary gastric cancer tissues. Slot blot hybridization was also performed to detect the amplification of EGFR gene. Immunohistochemically, 70 (42%), 87 (52%), 68 (41%) of 167 primary gastric cancers were stained positively for EGF, TGFalpha, and EGFR, respectively. Tumors with synchronous expression of EGFR and its ligands were most frequent in the following clinicopathological groups: tumors greater than 6 cm in size, those of the infiltrating type, and those of the poorly differentiated type. Among poorly differentiated carcinomas, the synchronous expression of EGFR and its ligands was more frequent in the infiltrating gross type than in the localized type. EGFR gene amplification was found in 5 (4.9%) of 103 primary tumors. EGFR gene amplification was also observed more frequently in infiltrating gross type of poorly,differentiated adenocarcinomas, as compared to localized gross type. These results indicate that the autocrine mechanism through EGFR may play an important role in the progression of infiltrating gross type of poorly differentiated adenocarcinoma of the stomach.

Prediction of the prognosis of patients with gastric-cancer by erbb-2 tissue status and expression of proliferating cell nuclear antigen.

Correlation between erbB-2 tissue status and expression of proliferating cell nuclear antigen (PCNA) was studied in 164 gastric carcinomas, and these results were compared with the risk of metastasis and prognosis. Thirty-one (19%) tumors showe evidence of erbB-2 protein expression, and the PCNA index ranged from 8.2% to 77.2% (mean; 40%). erbB-2 protein positive tumors were most often encountered in the following clinicopathological groups: tumors with serosal invasion, lymph node metastasis, and venous invasion. There was a significant correlation between PCNA index and lymph node metastasis. In addition, erbB-2 positive tumors showed significantly higher PCNA index than erbB-2 negative tumors. Cox prortional hazard model identified lymph node metastasis. In invasion, liver metastasis, and erbB-2 tissue status as independent prognostic factors, but PCNA index was not an independent prognostic factor. However, by combining erbB-2 tissue status and PCNA index, PCNA index. was a good prognostic factor particularly in patients with erbB-2 negative tumors. These results indicate that the determination of erbB-2 tissue status, when combined with PCNA index, may be useful in the prediction of the prognosis and in deciding the therapeutic schedule of patients with gastric cancer.

Chicken leg muscle alpha-connectin as studied by a monoclonal antibody to the 1200 kDa fragment.

Chicken leg gracilis muscle contained only alpha-connectin (ca 3000 kDa) without beta-connectin. When myofibrils were kept standing for 20 hr at 4 degrees C, alpha-connectin was degraded to beta-connectin (ca 2000 kDa) and 1200 kDa peptide. The latter was prepared from myofibrils and purified by gel filtration in the presence of SDS. A monoclonal antibody, alpha 7, to this 1200 kDa fragment was prepared. The antibody reacted with the 1200 kDa fragment and its mother molecule alpha-connectin, but not with beta-connectin. Immunoelectron microscopy using alpha 7, as well as other antibodies to chicken breast muscle beta-connectin, revealed that the 1200 kDa peptide covered the portion of alpha-connectin from the Z line to the N2 line region in the I band of chicken leg gracilis muscle sarcomeres. The results were in good agreement with those observed in rabbit skeletal muscle.