Chromosome condensation in Xenopus mitotic extracts without histone H1.

The contribution of histone H1 to mitotic chromosome condensation was examined with the use of a cell-free extract from Xenopus eggs, which transforms condensed sperm nuclei into metaphase chromosomes. When H1 was removed from the extract, the resultant metaphase chromosomes were indistinguishable from those formed in complete extract. Nucleosomal spacing was the same for both. Thus, H1 is not required for the structural reorganization that leads to condensed metaphase chromosomes in this egg extract.

Stimulation by thyroid-stimulating hormone and Grave's immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo.

To elucidate the pathogenesis of thyroid gland hypervascularity in patients with Graves' disease, we studied the expression of mRNAs for vascular endothelial growth factor (VEGF) and its receptor, Flt family, using human thyroid follicles in vitro and thiouracil-fed rats in vivo. Human thyroid follicles, cultured in the absence of endothelial cells, secreted de novo-synthesized thyroid hormone in response to thyroid-stimulating hormone (TSH) and Graves' IgG. The thyroid follicles produced VEGF mRNA but not flt-1 mRNA. The expression of VEGF mRNA was enhanced by insulin, tumor-promoting phorbol ester, calcium ionophore, dibutyryl cAMP, TSH, and Graves' IgG. When rats were fed thiouracil for 4 wk, their serum levels of TSH were increased at day 3. VEGF mRNA was also increased on day 3, accompanied by an increase in flt family (flt-1 and KDR/ flk-1) mRNA expression. These in vitro and in vivo findings suggest that VEGF is produced by thyroid follicles in response to stimulators of TSH receptors, via the protein kinase A and C pathways. VEGF, a secretable angiogenesis factor, subsequently stimulates Flt receptors on endothelial cells in a paracrine manner, leading to their proliferation and producing hypervascularity of the thyroid gland, as seen in patients with Graves' disease.

Remodeling of sperm chromatin induced in egg extracts of amphibians.

Sperm nuclear basic proteins of Bufo japonicus consist of 2 distinct protamines, whereas those of Xenopus laevis consist of 6 sperm-specific basic proteins (SP1-6) in addition to H3, H4 and smaller amounts of H2A and H2B. Cloning of pertinent cDNAs and partial amino acid sequence studies suggested that these 6 sperm-specific proteins of Xenopus are encoded by 3 distinct genes. Despite differences in their initial compositions of chromatin, sperm nuclei exposed to amphibian egg extracts rapidly decondense, lose sperm-specific basic proteins, and concomitantly form an ordinary nucleosome core consisting of H2A, H2B, H3, H4, and cleavage-stage specific subtype H1X. In this remodeling process, nucleoplasmin plays dual roles as a molecular chaperone, selectively removing sperm-specific basic proteins from, and bringing H2A and H2B to, sperm DNA. Thus remodeling of chromatin is induced even in mammalian (human) sperm nuclei under defined conditions including nucleoplasmin and exogenous histones.

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells.

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.

Endothelin-3 immunoreactivity in gonadotrophs of the human anterior pituitary.

Endothelin (ET), originally discovered in vascular endothelial cells, has also been demonstrated in nonvascular tissues. The present study was undertaken to elucidate the presence of ET in the human pituitary. The avidin-biotin complex method with antiserum to ET-1 (and ET-2) or ET-3 was used to identify ET in human pituitaries obtained by autopsy. ET-3 immunoreactivity was found in the cytoplasm of large ovoid cells of the anterior pituitary. Using the double staining method, the cells containing ET-3 immunoreactivity were differentiated from cells containing ACTH, TSH beta, GH, PRL, and protein S-100. By staining with anti-LH beta antiserum in adjacent sections and using the double staining method, the cells were identified as gonadotrophs. No staining was observed in the posterior pituitary. In addition, no ET-1 (and ET-2) immunoreactivity was detected. The specific localization of ET-3 immunoreactivity in the gonadotrophs of the human pituitary suggests a possible role of ET-3 in the regulation of anterior pituitary function.

Identification and analyses of the Xenopus TERT gene that encodes the catalytic subunit of telomerase.

The Xenopus telomerase catalytic component gene, xTERT (Xenopus telomerase reverse transcriptase), has been cloned. The production of xTERT recombinant protein together with the proposed Xenopus telomerase RNA (xTR) (Chen et al., 2000. Cell 100, 503-514) in a rabbit reticulocyte lysate system led to the reconstitution of active telomerase, indicating that both products are functional telomerase components. Both xTERT expression and telomerase activity are high from the early to the late blastula stage. However, they are decreased at the gastrula stage and thereafter, suggesting that the xTERT expression level is the primary mechanism for regulating telomerase activity in Xenopus development. This is the first report of a non-mammalian vertebrate TERT gene. Sequence comparison of xTERT with human and mouse TERTs has uncovered four regions conserved in the amino-terminal halves of vertebrate TERT proteins, the functions of which will be discussed herein.

Cloning and characterization of Aspergillus nidulans vpsA gene which is involved in vacuolar biogenesis.

In Saccharomyces cerevisiae, vacuoles play very important roles in pH and osmotic regulation, protein degradation and storage of amino acids, small ions as well as polyphosphates. In filamentous fungi, however, little is known about vacuolar functions at a molecular level. In this paper, we report the isolation of the vpsA gene from the filamentous fungus Aspergillus nidulans as a homologue of the VPS1 gene of S. cerevisiae which encodes a dynamin-related protein. The vpsA gene encodes a polypeptide consisting of 696 amino acids that is nearly 60% homologous to the S. cerevisiae Vps1. Similar to Vps1, VpsA contains a highly conserved tripartite GTPase domain but lacks the pleckstrin homology domain and proline-rich region. The vpsA disruptant shows poor growth and contains highly fragmented vacuoles. These results suggest that A. nidulans VpsA functions in the vacuolar biogenesis.

Novel combretastatin analogues effective against murine solid tumors: design and structure-activity relationships.

A series of combretastatin A-4 (CA-4) analogues were synthesized, and their cytotoxic effects against murine Colon 26 adenocarcinoma and inhibitory activity on tubulin polymerization were evaluated. Since CA-4 has limited aqueous solubility, the target compounds were designed to improve solubility by introduction of a nitrogen-containing group. Among the compounds synthesized, those with an amino moiety in place of the phenolic OH of CA-4 showed potent antitubulin activity and cytotoxicity against murine Colon 26 adenocarcinoma in vitro. Some of the compounds which were potent in vitro were evaluated in the murine tumor model Colon 26 in vivo. Among these, 13bHCl, 21aHCl, and 21bHCl showed significant antitumor activity in the animal model, while CA-4 was ineffective. 13bHCl and 21aHCl were further evaluated in two murine tumor models (Colon 38 and 3LL) and human xenografts HCT-15. These compounds showed potent antitumor activity comparable or superior to that of CDDP. The structure-activity relationships of this series of compounds are also discussed.

Cloning and nucleotide sequence analysis of immunodominant heat-shock protein of Yersinia enterocolitica.

Yersinia enterocolitica, a highly antigenic 60-kDa protein, designated cross-reacting protein antigen (CRPA), is a member of the chaperonin-60 family of molecular chaperones. The gene encoding CRPA was cloned, expressed and sequenced. A partial library from Y. enterocolitica 0:3 genomic DNA was constructed in vector pUC19 and was screened by the immunoreactivity to monoclonal antibody 1A4, which has specificity for a species-specific epitope on the CRPA molecule. The crpA gene region consists of a putative two-cistron operon encoding proteins of 549 and 97 amino acids. The operon structure was led by a consensus heat-shock promoter sequence. Homology searches using the derived amino acid sequence have revealed that CRPA is 88.2% identical to GroEL of Escherichia coli. CRPB, another protein encoded by the operon, shows extensive sequence homology, 91.8% identical to GroES of E. coli which is a member of chaperonin-10.

Potentiation of antitumor and antimetastatic activities of adriamycin by a novel N-alkylated dihydropyridine, AC394, and its enantiomers in colon cancer-bearing mice.

PURPOSE: We have previously shown that a series of N-alkylated 1,4-dihydropyridines potentiate the therapeutic efficacy of vincristine in vincristine-resistant P388 leukemia. The purpose of this study was to investigate the ability of one of the compounds, AC394, and its enantiomers to potentiate the antitumor activity of adriamycin against colon cancer cells in vitro and in vivo. METHODS: The effects of AC394 on potentiation of adriamycin cytotoxicity and enhancement of its accumulation were evaluated using colon 26, HCT-15 and MCF-7 cells. Furthermore, the activities of AC394 and its enantiomers were compared. We also studied the combined effects of (+)-AC394 and adriamycin on subcutaneously (s.c.)-implanted and liver metastasis tumor models. RESULTS: AC394 potentiated the cytotoxicity of adriamycin and enhanced its accumulation in colon cancer cells (colon 26 and HCT-15), which are known to express P-GP (P-glycoprotein) intrinsically. Enhancement of adriamycin accumulation by AC394 was found in s.c.-implanted colon 26 cells in vivo. Although both enantiomers of AC394 showed equal activity in vitro, (+)-AC394 was more effective than (-)-AC394 given orally. (-)-AC394 was found to be cleared more rapidly from the plasma than (+)-AC394. Thus, (+)-AC394 was evaluated for further study. Administration of (+)-AC394 significantly potentiated the antitumor activities of adriamycin in human colon cancer HCT-15 cells implanted s.c. Furthermore, in the liver metastasis model using colon 26 cells, a model completely resistant to adriamycin, the combination therapy of adriamycin with (+)-AC394 produced superior antitumor effects over adriamycin alone. CONCLUSIONS: A newly synthesized N-alkylated 1,4-dihydropyridine derivative, (+)-AC394, showed superior effects on the potentiation of adriamycin antitumor and antimetastatic activities in vivo. These results suggest that this combination may have therapeutic efficacy not only against primary colon cancers but also against metastatic liver cancer.

Do the substance P-ergic vagus afferents in cats reinnervate the deafferented superior cervical ganglion?

By ligating specific nerves in the cat, the central processes of the nodose ganglion (NG) cells containing substance P-like immunoreactive materials were shown, immunohistochemically, to run through the cross-anastomosis made between the cranial end of the NG and the caudal end of the superior cervical ganglion (SCG), and to reach the portions cranial to the SCG without making synaptic contacts with the SCG cells.

Effect of spinal ganglionectomy on substance P-like immunoreactivity in the gastroduodenal tract of cats.

Bilateral ligation of the splanchnic nerves and spinal ganglionectomy from T5 to L2 both produced a profound decrease of substance P (SP)-like immunoreactivity in Auerbach's plexuses of the duodenum and the antrum of the stomach in the cats. As unilateral dorsal rhizotomy from T5 to L2 produced no such changes, SP is probably synthesized in the spinal ganglion and transported through the splanchnic nerve to the gastroduodenal wall.

Density of Helicobacter pylori infection in patients with peptic ulcer perforation.

BACKGROUND: A lack of change in prevalence of severe ulcer complications requiring emergency operation has been reported, despite the common use of histamine-2 (H2)-receptor antagonists and proton pump inhibitors. This may be attributable to use of ulcerogenic drugs or Helicobacter pylori (HP) infection, or both. In this study, HP infection was evaluated semiquantitatively in patients with peptic ulcer who required surgery, and the severity of histologic change was investigated. METHODS: We reviewed a total of 113 consecutive patients (98 men and 15 women) operated on for perforation, hemorrhage, or stenosis of gastroduodenal ulcer between January 1986 and December 1995. Detection of HP was carried out by immunohistochemical staining. We graded the density of HP infection according to the number of individual HP bacteria counted in a highly magnified visual field (x 1,000 of light microscopy). The grade of HP infection was defined as follows: (0) = 0; (1+) = 1-9; (2+) = 10-29; (3+) = 30-99; (4+) > or = 100. The severity of gastritis was evaluated by histologic examination using the criteria of Rauws. RESULTS: Although the number of operations for gastroduodenal ulcer declined significantly, the rate of emergency operation for gastroduodenal ulcer increased from 60% to 90%, with the result that the frequency of operations for perforation or bleeding remained virtually constant and that for stenosis significantly decreased. HP infection was more prevalent in perforated ulcer (92%) than hemorrhagic ulcer (55%) or stenotic ulcer (45%). The grades of HP infection were 3.0 +/- 0.14 (mean +/- SEM) in perforated ulcer, 2.3 +/- 0.34 in hemorrhagic ulcer, and 2.5 +/- 0.22 in stenotic ulcer. Perforated ulcer was associated with significantly more severe HP infection and gastritis changes than hemorrhagic ulcer or stenotic ulcer. CONCLUSIONS: This study indicates that patients with perforated ulcer were infected with HP more severely than those with hemorrhagic ulcer or stenotic ulcer at the time of surgery. A close relationship was observed between the perforated ulcer and the density of HP infection determined semiquantitatively using immunohistochemical stain.

Jejunal pouch to avoid stricture after esophagojejunostomy with circular stapler.

BACKGROUND: Anastomotic stricture is one of the most common problems in esophagojejunostomy using the end-to-end anastomosing (EEA) instrument (Auto Suture Co, Norwalk, CT) after total gastrectomy. To alleviate the stricture, several methods, such as incision to the scar, balloon dilatation, and steroid injection are available. To avoid stricture, the jejunal pouch may allow use of a larger EEA than Roux-en-Y (ReY) reconstruction does. STUDY DESIGN: A total of 45 patients underwent curative total gastrectomy and esophagojejunostomy with jejunal pouch construction (27 patients) or ReY (18 patients), using the EEA. The effects of jejunal pouch construction with a large EEA on avoidance of stricture and benefit to nutritional status were investigated by comparing it with the ReY in terms of postoperative morbidity, postprandial symptoms, and nutritional parameters (serum protein, serum albumin, body weight). RESULTS: EEA28 or larger could be used in 25 patients in the pouch group and 8 patients in the ReY group (p < 0.05). Stricture developed in one patient in the pouch group and in four patients in the ReY group (p < 0.05). Postprandial symptoms were experienced less frequently (p < 0.05) in the pouch group than in the ReY group. When stricture and symptoms were analyzed according to the size of EEA, they occurred more frequently (p < 0.05) in the patients with EEA25 than those with EEA28 or EEA31. No significant differences were evident in nutritional parameters. CONCLUSIONS: The choice of jejunal pouch technique allowed the use of a larger EEA than that of ReY reconstruction, resulting in avoidance of anastomotic stricture and postprandial symptoms, though little benefit in nutritional status was evident to the patients after total gastrectomy.

Effects of MDR1/P-glycoprotein expression on prognosis in advanced colorectal cancer after surgery.

Resistance to chemotherapeutic agents is a major problem for successful cancer treatment. P-glycoprotein (Pgp), a product of the multidrug resistance (MDR)1 gene expressed in cancer cells, is one of the mechanism of MDR. However, there are few reports regarding the effects of Pgp on prognosis of colorectal cancer (CRC) after surgery. We examined a total of 80 patients (45 males and 35 females with an average age of 69 years) whose CRCs were classified into stage 2-4 and completely resected surgically in our institute between January 1990 and September 1999. To evaluate Pgp expression in CRC, immunohistochemical stain was performed with a monoclonal antibody. Relationships between Pgp expression and clinicopathological variables which may have affected prognosis were evaluated. Survival curves were calculated using the Kaplan-Meier method, and differences were evaluated with the log-rank test. The Cox's proportional hazards model was used in the univariate and multivariate survival analysis. Pgp expression showed a significant correlation with histological differentiation (p=0.023). However, no correlation was observed with gender, tumor location, lymph node metastasis, lymphatic invasion, venous invasion, and cancer stages. Survival rates after surgery tended (p=0.093) to be higher in Pgp (+) than Pgp (-) patients. Pgp was not a significant prognostic factor by univariate analysis and multivariate analysis adjusted for other clinicopathologic variables. Survival rates after surgery tended to be higher in Pgp (+) than Pgp (-) patients and Pgp expression was correlated with histological differentiation of CRC. Thus, a relative resistance of CRC to conventional chemotherapy may be partly caused by Pgp expressed in well or moderately differentiated CRC. However, Pgp expression was not a significant independent prognostic factor in advanced CRC after surgery.

Endothelin-1 production by human synoviocytes.

Immunoreactive (ir)-endothelin (ET)-1 concentrations in serum samples and synovial fluids from patients with rheumatoid arthritis were higher than concentrations in sera obtained from healthy volunteers. No significant difference in ir-ET-1 concentrations in synovial fluid was observed between rheumatoid arthritis patients and osteoarthritis patients. Cultured fluids of synovial cells collected from synovial tissues and leucocytes from synovial fluids of rheumatoid arthritis patients were studied to determine the origin of ir-ET-1 in synovial fluids. Ir-ET-1 was detected in the cultured fluids of synovial macrophage-like type A cells, but not in those of fibroblast-like type B cells from the synovial tissues or leucocytes from the synovial fluids. Longitudinal studies showed that the ir-ET-1 concentration in the cultured fluid reached a peak around 24 h after starting the culture. ET-1 secreted from macrophage-like synoviocytes may be involved in the pathogenesis of inflammatory arthritis.

Postsurgical sequential methotrexate/5-FU and leucovorin on outpatient basis for advanced colorectal carcinoma.

BACKGROUND/AIMS: The present study compared the effects of sequential methotrexate and 5-fluorouracil followed by leucovorin rescue (MFL), as an adjuvant chemotherapy versus a combination of UFT and mitomycin C (MMC), on patient survival and recurrence after surgery for colorectal carcinoma. METHODOLOGY: Between January 1990 and December 1997, a total of 55 patients with advanced colorectal cancer were treated postsurgically by adjuvant chemotherapy using MFL or UFT-MMC. Surgical treatment was performed according to standardized procedures for radical resection of colorectal cancer. The patients were divided into 2 groups after surgery. The MFL regimen consisted of MTX (100 mg/m2) and 5-FU (600 mg/m2) at hour 24, followed by leucovorin rescue. The UFT-MMC regimen consisted of MMC (12 mg/m2) intraoperatively and MMC (6 mg/m2) every other week after surgery for 2 months, and oral UFT (375 mg/m2/day), a combination of tegafur and uracil in a molar ratio of 1:4, was continued for 3 years or longer depending on the patients tolerance. RESULTS: The overall survival rates after surgery were significantly (P < 0.05) higher in the MFL than the UFT-MMC group. Recurrence rates were significantly lower in the MFL than the UFT-MMC group, especially for liver recurrence. Disease-free survival was significantly (P < 0.05) higher in the MFL than the UFT-MMC group. CONCLUSIONS: These results demonstrated the superiority of MFL therapy for improving postsurgical survival in patients with advanced colorectal cancer, in particular those patients with a high risk of recurrence following potential curative resection.

Surgical risks of acute cholecystitis in elderly.

BACKGROUND/AIMS: For the elderly patient, an emergency biliary procedure carries a higher risk than an elective operation. Recently introduced advances in ultrasonography and critical care medicine have affected the clinical risks of surgery for acute cholecystitis in the elderly. This study evaluated the clinical risks of open cholecystectomy for the elderly with acute cholecystitis. MATERIALS AND METHODS: During a 10 year period (1985-1994), a total of 145 patients were diagnosed with acute cholecystitis and underwent cholecystectomy. According to their age, the patients were divided into 3 groups (Group A < 59 years of age; Group B between 60-69 years of age; Group C > 70 years of age). The characteristics and the surgical risk factors in open cholecystectomy for the elderly with acute cholecystitis were evaluated. RESULTS: The rate of acalculous cholecystitis and choledochal stones were significantly (p < 0.05) high in Group C. Septic complication, gangrenous changes, and positive bile culture were also increased parallel to the increase in age. A noteworthy finding was an incidental carcinoma found in a case in group B and in 3 cases in group C. Hospital stay was significantly longer in Group C than in the other groups due to pre-operative complications and post-operative morbidity. CONCLUSION: With respect to increase in elderly patients with acute cholecystitis who present more frequent gangrenous changes and carcinomatous changes as well as high rate of septic complication, successful treatment of these patients is increased when early surgery can be performed on the basis of accurate and prompt diagnosis using imaging modalities and meticulous peri-operative management.

A case of colon cancer recurrence with P-glycoprotein treated by methotrexate, fluorouracil, and leucovorin.

A 68 year-old female underwent right hemicolectomy for an advanced cecum cancer and had been well without any evidence of recurrence for a year after surgery. Despite post-operative treatment with oral Tegafur (400 mg/m2/day), CEA level increased gradually beginning 15 months after surgery. Sequential chemotherapy with methotrexate (MTX) and 5-Fluorouracil (5-FU), followed by leucovorin rescue (MFL) was started on an outpatient basis, and has been continued every 4 weeks since then. It consisted of MTX (100 mg/m2) and 5-FU (600 mg/m2) started 24 hours after MTX, followed by oral leucovorin (15 mg/body) started 30 hours after MTX 6 times at intervals of 6 hours. CEA level declined initially, but increased slowly for 3 years on MFL, although no evidence of recurrence was detected by imaging studies with computed tomography, ultrasound, and scintigram. Four years after surgery, a tumor recurrence developed in the abdominal wall. The patient underwent resection of the tumor, resulting in a decline of the CEA level. She has been alive and well for 5 years on MFL after the primary surgery. Both the original tumor and recurrent tumor showed immunoreactivity for P-glycoprotein. The present case demonstrates the feasibility of using MFL on an outpatient basis, and its potential to suppress the colon cancer growth with P-glycoprotein expression.

Sigmoid colon diverticulosis adherent to mesh plug migration after open inguinal hernia repair.

PURPOSE: Tension-free hernia repair with polypropylene mesh plug and patch is currently the most popular technique for open inguinal hernioplasty. It is well tolerated by most patients with few complications. Despite these excellent results, late-onset complications may occur. METHODS: An 83-year-old man reported 2 weeks of bloody stool. His medical history was significant for a left open inguinal herniorrhaphy with the mesh plug and patch technique. Barium enema revealed a stenotic segment in the sigmoid colon and multiple diverticulosis. Because a malignant lesion could not be ruled out, the patient underwent an operation. Laparotomy revealed an inflamed sigmoid colon with diverticulosis adherent to a hard tumor, which was mesh plug used for the previous open inguinal hernia repair. After mobilization of the adhesion between the mesh plug tumor and the sigmoid colon, sigmoidectomy was performed. The patient's postoperative course was uneventful. CONCLUSIONS: We reported a case of sigmoid colon diverticulosis adherent to mesh plug migration after open inguinal hernia repair. The potential risk of plug migration should be well understood by the surgeon. To avoid this risk completely, several methods have been proposed such as suturing the plug and patch together, or using an all-in-one design such as the Prolene Hernia System (Johnson and Johnson Co., Tokyo, Japan).