Isolation and partial purification of a new class of transforming growth factors from an avian sarcoma virus-transformed rat cell line.

Transformation of rat cells by avian sarcoma viruses induced the release of growth factors into serum-free conditioned medium. An avian sarcoma virus-transformed rat cell line, 77N1, produced and released a polypeptide growth factor, classified as a transforming growth factor (TGF), which transiently promotes anchorage-dependent BALB3T3 A31 cells to form progressively growing colonies in soft agar. The TGF was isolated and partially purified from an extract of 77N1 cells by ion-exchange chromatography on a diethylaminoethyl Sephacel column followed by ammonium sulfate precipitation. The TGF was assumed to have a molecular weight of 11,000 from gel filtration on Sephadex G-50. This TGF did not compete with epidermal growth factor for binding to cell membrane receptors and was not potentiated by epidermal growth factor. The TGF was trypsin and dithiothreitol sensitive as well as heat and acid labile, indicating that it was different from previously reported TGFs of similar molecular weight and thus belonged to a new class of TGFs.

Slow-Growing Large Irritation Fibroma of the Anterior Hard Palate: A Case Report Using Immunohistochemical Analysis.

Irritation fibromas are recognized as fibrous lesions, usually reactive hyperplasias; however, the mechanism of enlargement is unclear. This paper reports on an abnormally large irritation fibroma of extremely gradual growth. The immunohistochemical features (CD34, α-SMA, vimentin, Ki-67, and TGF-α) of this irritation fibroma are presented to distinguish reactive hyperplasia from other true fibrous neoplasm diseases. In the only previous study, it was reported that the expression of TGF-α might be associated with the development of oral fibromas. Therefore, we investigated the relationship between this exceptionally-large fibrous lesion of extremely slow growth and the immunohistochemical reactivity of TGF-α, finding that, in contrast to the previous study, TGF-α was not expressed. This is the first study to evaluate the enlargement mechanism of such a large irritation fibroma using the approach of immunohistochemical analysis, and it indicates that such analysis can help elucidate the diverse causes and enlargement mechanisms of irritation fibromas.

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture.

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.

Inhibition of different steps of the ubiquitin system by cisplatin and aclarubicin.

Ubiquitin is involved in such fundamental cellular processes as cell cycle control, DNA repair, protein degradation and stress responses. We previously reported that cisplatin could inhibit the ubiquitin-ATP-dependent proteolysis and ubiquitination. We further investigated the effect of various antitumor agents on the ubiquitin system and found that aclarubicin (ACR) inhibits the ubiquitin-ATP-dependent proteolysis but not the ubiquitination process. We found that ACR as well as cisplatin inhibited the ubiquitin-ATP-dependent proteolytic activity of rabbit reticulocytes. The IC50 values of these agents were 52 and 90 microM, respectively. Although cisplatin inhibits the conjugation of ubiquitin to proteins through the inhibition of a ubiquitin-activating enzyme, ACR, at 120 microM, does not. Thus, the antitumor agents affecting the ubiquitin system could be classified into two groups; one is represented by cisplatin, which inhibits the ubiquitination of the proteins, and the other is ACR, which does not inhibit the ubiquitination but does inhibit the ubiquitin-ATP-dependent proteolysis. Mitomycin C belongs to the latter group.

Reversal of neuronal polarity characterized by conversion of dendrites into axons in neonatal rat cortical neurons in vitro.

The mechanisms for the establishment and maintenance of cell polarity in neurons are not well understood. Axon regeneration from dendrites has been reported after axotomy near the cell body in vivo. We report here in vitro a reversal of neuronal polarity characterized by the conversion of dendrites into axons. We isolated neurons from the neonatal rat cerebral cortex. Neurons that exhibited an apical dendrite with a length of >100 microm were monitored for 3 days in culture. In 66% of neurons examined, a new axon, as identified by reactivity with an antibody to dephosphorylated tau or by lack of reactivity with an antibody to the a and b isoforms of microtubule-associated protein 2, appeared to form from the tip of the original dendrite. Further analysis of such neurons revealed that the distal half of the original dendrite became positive for dephosphorylated tau or negative for microtubule-associated protein 2. Time-lapse video microscopy demonstrated the conversion of the original dendrite into an axon without dendritic retraction. Axon regeneration from dendritic tips required a significantly longer time than axon regeneration from minor processes. Our observations thus demonstrate in vitro a time-consuming reversal of neuronal polarity and the conversion of a dendritic cytoskeleton into an axonal one.

Microsatellite instability correlates with normal expression of cyclin E in hepatocellular carcinomas.

It has been reported that microsatellite instability (MSI) strongly correlates with carcinogenesis and cancer progression. In the present study, we studied the incidence of MSI at 5 polymorphic microsatellite markers (D5S406, D13S153, D16S402, D17S796, and poly(A) tract BAT26), the expression of G1 cyclins (cyclin A, cyclin D and cyclin E), and Ki-67 labeling index in 30 surgically resected hepatocellular carcinomas (HCCs) and their adjacent non-cancerous tissues. The results of analysis showed that 43% of HCCs exhibited MSI in one locus, 10% in two loci, and 3% in three loci. Overexpressions of cyclin E and cyclin A were observed in 57% and 83% of HCCs, respectively. MSI in HCCs, however, correlated with normal expressions of cyclin E and cyclin A and with a low labeling index of Ki-67. Thus, patients with HCCs exhibiting MSI at these 5 markers may have less involvement of G1/S disregulation and may have better prognosis than other patients with HCC.

Different expression of positive and negative regulators of hepatocyte growth in growing and shrinking hepatic lobes after portal vein branch ligation in rats.

Portal vein branch embolization is often performed before hepatectomy to prevent postoperative liver failure. It is, however, still not clear how the embolized lobe shrinks and the non-embolized lobe proliferates in counterbalance. We investigated the expression of positive and negative regulators of hepatocyte growth to clarify the mechanisms of liver growth and atrophy in a rat portal vein ligation (PVL) model compared with partial hepatectomy (PH). A significant increase in DNA synthesis within the non-ligated lobe reached a peak at 36 h, a delay of 12 h as compared with PH, while no increase occurred in the ligated lobe. Expression of hepatocyte growth factor mRNA remarkably increased in the non-ligated growing lobe between 6 and 24 h, but was only slightly elevated in the ligated shrinking lobe. Contrarily, negative regulators of hepatocyte proliferation, such as TGF-beta1 and IL-1beta, were strongly expressed in the ligated shrinking lobe. Thus, the changes of portal venous flow and/or pressure caused by PVL may contribute to induction of different kinds of growth factors between the ischemic and non-ischemic lobes; these factors possibly regulate liver regeneration and atrophy after PVL.

Inhibition of ubiquitin-ATP-dependent proteolysis and ubiquitination by cisplatin.

We tested the inhibitory activity of various antitumor agents on the ubiquitin-ATP-dependent proteolytic activity in rabbit reticulocyte lysates. We found that cisplatin, 4'-(9-acridinyl-amino) methanesulfon-m-anisidide (m-AMSA) and mitomycin C inhibited the ubiquitin-ATP-dependent proteolysis. IC50 values (50% inhibition concentrations) of these antitumor agents were 90, 210 and above 290 microM, respectively. Furthermore, cisplatin was found to inhibit the conjugation of ubiquitin to endogenous proteins in fraction II at 100 and 330 microM. These results suggest that cisplatin interacts with the enzyme(s) involved in ubiquitin conjugation and thus inhibits the ubiquitin-ATP-dependent protein degradation. We assume that the agents that can affect the ubiquitin system might be useful for the treatment of tumors and that the ubiquitin system could be a new target for cancer chemotherapy.

Enhanced expression of cyclin E and cyclin A in human hepatocellular carcinomas.

BACKGROUND: Disruption of the G1/S check point leads to uncontrolled cell growth, resulting in the development of cancers. MATERIALS AND METHODS: Cell cycle regulatory molecules were investigated in 33 surgically resected hepatocellular carcinoma (HCC) samples from 30 patients by Western blotting. RESULTS: Enhanced expressions of cyclin E and cyclin A were detected at frequencies of 18/33 and 26/33 in HCCs, respectively, as compared with their neighboring noncancerous tissues. The enhanced expression of cyclin E, but not that of cyclin A, correlated with hyperphosphorylation of pRb and high frequency of Ki-67-positive cells. Thus, the HCCs with enhanced cyclin E expression probably contain a relatively large number of proliferating cancer cells. CONCLUSIONS: The degree of cyclin E expression can be used as a prognostic parameter of HCC. In addition, cyclin E may become a molecular target in the treatment of HCCs.

Establishment of a human hepatoma cell line, HLE/2E1, suitable for detection of p450 2E1-related cytotoxicity.

By transfection of an expression vector of human cytochrome P450 2E1 (CYP2E1) into a human hepatoma cell line (HLE), a new cell line (HLE/2E1) that stably expresses activity of CYP2E1 has been established. The HLE/2E1 cell line expressed a higher level of CYP2E1 messenger ribonucleic acid than did the mother HLE cell line. CYP2E1 enzyme activity determined by a p-nitrophenol oxidation assay was also higher in HLE/2E1 cells than in HLE cells. In addition, the enzyme activity of the HLE/2E1 cells was increased by ethanol treatment. Exposure to acetaminophen (APAP) or buthionine sulfoximine (BSO) caused a greater decrease in viability of the HLE/2E1 cells than that of the HLE cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The cytotoxicity of APAP or BSO to HLE/2EI cells was inhibited by the addition of ethanol or vitamin E. However, the cytotoxicity of both APAP and BSO was enhanced by 24-h preincubation of HLE/2E1 cells with ethanol. These results show that this cell line provides a useful model for studying catalytic properties of CYP2E1 and cytotoxic mechanisms of chemicals metabolized by CYP2E1.

Superior mesenteric artery syndrome in identical twin brothers.

We report two identical male twins who suffered from superior mesenteric artery (SMA) syndrome. A 28-year-old man was admitted for investigation of postprandial nausea and vomiting. Upper gastrointestinal examination revealed a dilated proximal duodenum with an abrupt vertical cutoff of barium flow in the third portion of the duodenum, establishing the diagnosis of SMA syndrome. One year later, his twin brother also presented similar symptoms and was radiologically diagnosed as SMA syndrome. The twin brothers did not respond adequately to conservative therapy and underwent duodenojejunostomy. This is the first report of SMA syndrome in identical twins.

Sigmoid colon obstruction due to blunt abdominal trauma: a case report.

Post-traumatic colonic stenosis (obstruction) is rare. We experienced a case of sigmoid obstruction due to blunt abdominal trauma. A 75-year-old man was hit on the lower abdomen 3 days before admission and gradually developed abdominal pain and distension. Laboratory data showed severe inflammation and a barium enema disclosed obstruction of the sigmoid colon. Conservative treatment was carefully carried out, because there was no sign of peritoneal irritation and there were passages of normal stool and flatus. The sigmoid obstruction gradually improved and the stenosis was almost undetectable on a barium enema on the 51st hospital day. An abdominal contusion is the most likely causal factor in this case. Compression of the sigmoid colon between the abdominal wall and the promontory of the pelvis is the most possible explanation.

Continuous measurement of tissue oxygen and carbon dioxide gas tensions in dog liver in ischemia/reperfusion.

An experiment was conducted to determine whether the oxygen and carbon dioxide gas tensions in liver tissue (PtO2 and PtCO2, respectively) reflect the state of microcirculation and/or metabolism in the ischemic liver. Subjects were divided into three groups: group 1, 30 min ischemia; group 2, 60 min ischemia; group 3, four times of intermittent 15 min ischemia after every 10 min of reperfusion. PtO2, PtCO2 and tissue blood flow (TBF) were measured by mass spectrometry, comparatively studied with the serum GOT level as an indicator of liver tissue damage. Furthermore, the time point at which the PtCO2 increase for 1 min initially became less than 1/2 of the maximum value was located on the transit curve of PtCO2, referred to as the critically anaerobic (CA) point, with which new indices of critically anaerobic score (CAS) and time (CAT) (see details in text) were developed. The profiles of PtO2 and PtCO2 during ischemia and reperfusion were clearly demonstrated, and the CA point was observed 12.7 +/- 2.9 min after induction of ischemia. PtO2 was positively correlated with TBF and negatively with the serum GOT level. Furthermore, not only CAS but also CAT were significantly correlated with PtO2, TBF, and the serum GOT level. It was concluded that PtCO2 reflects the state of anaerobic tissue metabolism during ischemia and PtO2 reflects the magnitude of microcirculatory disturbance and tissue injury caused by ischemia/reperfusion. Therefore, continuous monitoring of not only PtO2 but also PtCO2 is beneficial for patients undergoing hepatic surgery with ischemia.

[An immunoelectron microscopic study of distribution and density of nerve fibers innervating the rat iris].

We studied the sympathetic, parasympathetic and sensory nerve fibers in the rat iris using electron microscopic immunohistochemical techniques. Antibodies raised against tyrosine hydroxylase (TH), substance P (SP), and calcitonin gene-related peptide (CGRP) were used alone or combined together to characterize the nerve fibers. The ratios of the TH-positive, SP/CGRP-positive and TH/SP/CGRP-negative fibers were 18%, 37%, and 45% in the anterior half of the dilator stroma, 11%, 3%, and 86% in the posterior half of the dilator stroma, 45%, 26%, and 29% in the sphincter stroma, and 23%, 1%, and 76% in the sphincter muscle layer. These results confirm that the sympathetic and parasympathetic fibers doubly innervate both rat iris sphincter and iris dilator muscles, whereas sensory nerve fibers innervate mainly the stroma.

[Characterization of a transforming growth factor produced in rat cells transformed by an avian sarcoma virus].

Transformation of rat cells by avian sarcoma viruses (ASVs) induced the release of transforming growth factors (TGFs), which transiently promote anchorage-dependent untransformed BALB3T3 clone A31 cells to form progressively growing colonies in soft agar, into serum-free conditioned medium. A new class of TGF, which is a heat and acid-labile protein with a molecular mass of 12,000 daltons, was isolated and purified from an ASV-transformed rat cell line, 77N1, derived from NRK cells. The TGF from 77N1 did not bind to epidermal growth factor membrane receptors. We observed specific in vitro phosphorylation of several membrane proteins from A31 cells in the presence of the TGF.

[Suppression of postoperative liver metastases from colon cancer by continuous intraportal infusion of an angiogenesis inhibitor FR-118487].

A rabbit VX2 colon cancer model with spontaneous liver metastases was used to evaluate the antitumor effect of an angiogenesis inhibitor, FR-118487. FR-118487 (1 mg/kg/day) was infused continuously into the portal vein for a week after resection of primary colon cancer lesions (FR group). The incidence of liver metastases was 71.4% (5/7) in FR group, and 100% (7/7) in control group. The number and the weight of liver metastatic nodules were 31.0 +/0 36.0 and 1.4 +/- 1.8 g in FR group versus 83.7 +/- 73.9 and 6.5 +/- 4.9 g in control group, respectively. The metastases in FR group were significantly decreased in weight compared with those in control group (p < 0.05). No anastomotic leakage was recognized in either group. No side effects of FR-118487 such as body weight loss were found. Continuous intraportal infusion of FR-118487 in the early postoperative period may be effective to suppress liver metastases from colon cancer by inhibiting the angiogenesis concerning liver metastases.

A case of middle-ear cavernous lymphangioma with facial palsy.

OBJECTIVE: Only a few benign tumours of the middle ear have been reported to lead to the development of facial palsy. Here, we describe a patient with middle-ear cavernous lymphangioma and facial palsy. STUDY DESIGN: Single case study. PATIENT: A 61-year-old man presented with left-sided hearing impairment and incomplete left facial palsy. A tumour was confirmed to be occupying the epi- to mesotympanum and to be joined to the facial nerve. The tumour was removed along with facial nerve tissue, which was resected at its horizontal portion, and the remaining facial nerve was fixed by end-to-end anastomosis. Complete facial paralysis occurred after the operation, but the patient's House-Brackmann grade gradually improved to grade III. Post-operative histopathological examination revealed infiltration of the lymphangioma into the facial nerve tissue, together with mild neural atrophy of the facial nerve. CONCLUSION: These findings suggested that tumour invasion was the cause of facial palsy in this patient.