Internalization of a novel, huge lectin from Ibacus novemdentatus (slipper lobster) induces apoptosis of mammalian cancer cells.

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

The long-term outcome of transvaginal anterior levatorplasty for intractable rectovaginal fistula.

AIM: Several procedures have been described for rectovaginal fistula with a wide range of success, but there is little information on the long-term outcome. The aim of the present study was to investigate the long-term outcome after transvaginal anterior levatorplasty (ALP) for intractable rectovaginal fistula. METHOD: Data of 16 consecutive patients undergoing transvaginal ALP with fistulectomy and closure of the rectum and vagina between 1998 and 2011 were prospectively recorded and retrospectively investigated to study the long-term outcome. RESULTS: Birth injury (n = 7), low anterior resection for rectal cancer (n = 3), pouch surgery for ulcerative colitis (n = 2) and a procedure for prolapse and haemorrhoids (n = 2) were the main causes of the fistula. Nine patients had a covering stoma before surgery. All patients underwent ALP, with a covering stoma in two patients. Infection occurred in one patient and wound rupture after surgery in another patient. These patients underwent reoperation by ALP. All fistulae had healed at a median follow-up of 84 (8-193) months after initial surgery or stoma closure. CONCLUSION: Transvaginal ALP is effective for the treatment of mid or low rectovaginal fistula. The results show that a graft is not necessary regardless of whether or not previous surgery has been performed.

A theoretical and experimental study on meridional-facial isomerization of tris(quinolin-8-olate)aluminum (Alq3).

The rationale behind the stereospecific synthesis of a facial isomer of tris(quinolin-8-olate)aluminum (Alq3) is studied by density functional theory (DFT) calculations, which predict the favourable influence of an H3O(+) ion on the distribution ratio between a meridional and a thermodynamically unstable facial isomer.

Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease.

SBL/RC-RNase was originally isolated from frog (Rana catesbeiana) oocytes and purified as a novel sialic acid-binding lectin (SBL) that displayed strong anti-cancer activity. SBL was later shown to be identical to a ribonuclease (RC-RNase) from oocytes of the same species. The administration of SBL/RC-RNase induced apoptosis (with nuclear condensation and DNA fragmentation) in mouse leukemia P388 cells but did not kill umbilical vein endothelial or fibroblast cells derived from normal tissues. The cytotoxic activity of SBL/RC-RNase was inhibited by desialylation of P388 cells and/or the co-presence of free bovine submaxillary mucin. FACS analysis showed that SBL/RC-RNase was incorporated into cells after attachment to cholesterol-rich microdomains. Addition of the cholesterol remover methyl-ß-cyclodextrin reduced SBL/RC-RNase-induced apoptosis. Apoptosis occurred through the caspase-3 pathway following activation of caspase-8 by SBL/RC-RNase. A heat shock cognate protein (Hsc70) and a heat shock protein (Hsp70) (each 70 kDa) on the cell membrane were shown to bind to SBL/RC-RNase by mass spectrometric and flow cytometric analyses. Quercetin, an inhibitor of Hsc70 and Hsp70, significantly reduced SBL/RC-RNase-induced apoptosis. Taken together, our findings suggest that sialyl-glycoconjugates present in cholesterol-rich microdomains form complexes with Hsc70 or Hsp70 that act as triggers for SBL/RC-RNase to induce apoptosis through a pathway involving the activation of caspase-3 and caspase-8.

Short-term outcomes of local correction of stoma prolapse with a stapler device.

BACKGROUND: The aim of the present study was to classify the short-term outcomes of local correction of stoma prolapse with a stapler device. METHODS: The medical records of 11 patients undergoing local correction of stoma prolapse using a stapler device were retrospectively reviewed. RESULTS: No mortality or morbidity was observed after the surgery. Median operative time was 35 min (range 15-75 min), and blood loss was minimal. Median duration of follow-up was 12 months (range 6-55 months). One of the 11 patients had a recurrent stoma prolapse. CONCLUSIONS: This technique can be a feasible, safe and minimally invasive correction procedure for stoma prolapse.

Nodular-type muscle sarcoidosis developing after a dog bite.

We report a 46-year-old woman who experienced swelling of the right thigh around the wound caused by a dog bite. Physical findings, laboratory findings, imaging studies, and muscle biopsy showed that this was nodular-type muscle sarcoidosis. This is an unusual case of sarcoidosis, and the possibility is discussed that a dog bite may serve as a trauma to trigger sarcoidosis in genetically predisposed individuals.

Who can get the beneficial effect from lateral lymph node dissection for Dukes C rectal carcinoma below the peritoneal reflection?

PURPOSE: This study was designed to identify those patients with Dukes C rectal carcinoma below the peritoneal reflection who might benefit from lateral lymph node dissection. METHODS: The study involved 104 consecutive Dukes C patients who received total mesorectal excision with lateral lymph node dissection for rectal carcinoma below the peritoneal reflection between 1990 and 2002. The patients were retrospectively divided into three groups: patients without lateral spread (Group I: n = 52), patients with nodal involvement between the inferior hypogastric nerve and the internal iliac artery (Group II: n = 16), and patients with nodal involvement in the obturator space (Group III: n = 36). The patients also were divided into two groups according to the number of lateral nodes involved: less than four (n = 42) and at least four (lateral nodes involved: n = 10). Nodal involvement was determined histologically. RESULTS: The local recurrence and overall five-year survival rates were 5.8 and 66.9 percent in Group I, 18.8 and 59.8 percent in Group II, and 33.3 and 23.6 percent in Group III, respectively. These outcomes did not differ significantly between Groups I and II, but they were significantly worse in Group III than in Groups I and II, with the survival being significantly better in the patients with less than four histologically positive lateral nodes involved (43.2 percent) than in those with at least four positive lateral nodes involved (0 percent). CONCLUSIONS: Lateral lymph node dissection was effective for Dukes C rectal carcinoma below the peritoneal reflection with positive lateral nodes involved in the space between the autonomic nerve and the internal iliac artery and in patients with less than four positive lateral nodes.

Transvaginal anterior levatorplasty with posterior colporrhaphy for symptomatic rectocele.

BACKGROUND: We evaluated functional and morphological outcomes of transvaginal anterior levatorplasty with posterior colporrhaphy for symptomatic rectocele. METHODS: Ten women (median 68 years) underwent transvaginal anterior levatorplasty with posterior colporrhaphy for symptomatic rectocele. Symptoms and continence were monitored before and after surgery. Manovolumetric study and defecography were performed in 9 of 10 patients before and 3-6 months after surgery. Twenty-one females without anorectal diseases were used as controls in manovolumetric study. The patients were followed up after a median of 89 months (range, 3-103). RESULTS: Main symptoms (defecatory disorders in 9 patients, vaginal mass in 6, perineal discomfort in 2) disappeared after surgery. Six patients performed digitation preoperatively and gave up digitation on defecation after surgery. Stool incontinence disappeared in 4 of 5 preoperatively incontinent patients (Cleveland clinic score, 5-12) and continence score improved from 5 to 2 in the remaining patient. Three patients with urinary cough incontinence preoperatively did not experience incontinence after surgery but cough incontinence occurred occasionally in an 81-year-old patient postoperatively. Rectocele demonstrated on defecography disappeared postoperatively in all 9 patients who underwent defecography. High threshold volume and maximum tolerable volume, which were observed preoperatively, decreased to control levels after surgery. CONCLUSION: Transvaginal anterior levatorplasty with posterior colporrhaphy might be an option for symptomatic rectocele to improve anorectal and urinary dysfunctions with morphological disorders.

Minimally invasive transanal surgery for localized rectal carcinoid tumors.

Local excision is often fully justified for rectal carcinoid tumors. However insufficient surgical field and difficult access to proximal tumors have been drawbacks in performing pre-existing local excision procedures. A novel local excisional technique called minimally invasive transanal surgery (MITAS) has been experimented for local removal of carcinoid tumors in the rectum. A specially designed anal retractor connected to the Octopus retractor holder was used and an ENDO-stapler allowed the simultaneous excision and anastomosis to be performed. Eight patients with carcinoid tumors in the rectum (4 tumors in the upper rectum) underwent MITAS. Median distance from anal verge to proximal tumor was 6.5 cm (range, 5-12 cm). The median diameter of the tumor was 9 mm. Median operative time was 18.5 minutes and blood loss was minimal. No analgesics were needed postoperatively, and there was no morbidity or mortality. Full-thickness excision of the rectum was accomplished and the tumors confined in the submucosa were demonstrated histologically to be with free surgical margins. No recurrences have been observed with a median follow-up period of 39 months. The technique facilitates total excisional biopsy for rectal carcinoid tumors and reduces operative time, blood loss and complications.

Electroporation-mediated transfer of cytokine genes into human esophageal tumors produces anti-tumor effects in mice.

Electroporation facilitates transfer of chemicals or plasmid DNA from extracellular milieu into cells by increasing the permeability of the cell membrane. Delivery of electric pulses to established tumors thereby can improve the susceptibility of tumors to an anti-cancer agent administered. We examined whether electroporation-mediated transfer of cytokine genes into solid tumors could produce anti-tumor effects in the tumor-bearing mice. Plasmid DNA containing cytokine genes were injected into human esophageal T.Tn tumors developed in nude mice and electric pulses were then delivered. Administration of murine GM-CSF or human IL-2 gene followed by electroporation significantly suppressed the subsequent growth of T.Tn tumors and prolonged the survival of the inoculated mice. In contrast, electroporation-mediated introduction of a control gene, human GM-CSF gene, whose products do not bind to murine GM-CSF receptors, did not achieve any anti-tumor effects. In vivo transfection of cytokine genes with electroporation could be a possible therapeutic strategy for established solid tumors.

Identification of immunostimulatory DNA-induced genes by suppression subtractive hybridization.

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80-90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODN-stimulated bone marrow-derived macrophages: NF-kappaB p105, IRF-1, PA28beta, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms.

Combinatory anti-tumor effects of electroporation-mediated chemotherapy and wild-type p53 gene transfer to human esophageal cancer cells.

Delivery of electric pulses to an established solid tumor augments the permeability of cell membrane and increases the susceptibility of tumors to an anti-cancer agent that is administered in the vicinity of tumors. Forced expression of the wild-type p53 gene in tumor cells that have non-functional p53 gene(s) can also enhance their sensitivity to a DNA-damaging agent. To investigate the feasibility of electroporation-mediated therapy for cancer, electric pulses were delivered to human esophageal tumors developed in nude mice after they received an anti-cancer agent and/or plasmid DNA containing the wild-type p53 gene. The growth of esophageal tumors was suppressed with electroporation-mediated chemotherapy compared with the treatment with an anti-cancer agent or electroporation alone. Intratumoral injection of the wild-type p53 gene into p53-mutated esophageal tumors followed by electroporation also inhibited tumor growth. When mice were administered with the wild-type p53 gene and an anti-cancer agent, subsequent electroporation produced a synergistic therapeutic effect. Combinatory transfer of plasmid DNA and a pharmacological agent by electroporation is thereby a possible therapeutic strategy for the treatment of solid tumors.

Toxic shock syndrome toxin-1 accelerated collagen-induced arthritis in mice.

The aim of this study was to explore the roles of toxic shock syndrome toxin-1 (TSST-1) in collagen-induced arthritis (CIA). DBA/1 mice were immunized with type II collagen (CII) and treated with TSST-1. Intraperitoneal and intravenous injections of TSST-1 aggravated CIA, enhancing its incidence and severity. CIA was accompanied by an increase in anti-CII IgG Ab levels. Intraperitoneal administration with TSST-1 enhanced IFN-gamma, TNF-alpha, and IL-4 production in DBA/1 mice. We discovered the mRNA expressions of IFN-gamma, IL-2, TNF-alpha, IL-1beta, and iNOS in spleen cells stimulated with TSST-1 in vitro. However, IL-12 and IL-4 mRNA expression were seen constitutively without stimulation. Only a little increase of IL-12 and IL-4 mRNA expression was seen at 2-3 h after treatment with TSST-1. Our experiments demonstrated that CIA was aggravated by the treatment with TSST-1, which may have induced various proinflammatory cytokines and the production of both Th1 and Th2 cytokines.

Retrovirus-mediated gene transfer of granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction of their differentiation by G-CSF.

Myelodysplastic syndromes (MDS) are clonal disorders in which the proper differentiation of hematopoietic stem cells is impaired. There is no effective treatment for this stem cell disorder at present. In an attempt to find a new strategy that promotes the differentiation of MDS blast cells, we tried retroviral transduction of granulocyte colony-stimulating factor receptor (G-CSFR) into an interleukin-3-dependent MDS cell line, MDS-L, since expression of G-CSFR is known to be essential for the differentiation of myeloid progenitor cells and this expression is impaired in most MDS cells. Ectopic expression of human G-CSFR cDNA in MDS-L cells gave rise to granulocytic differentiation by G-CSF stimulation. G-CSF caused the transformants expressing G-CSFR to display a morphological characteristic of mature granulocytes, upregulated CD11b on the cell surface, and improved NBT reduction activity. These results demonstrate that MDS-L cells ecopically expressing G-CSFR are induced to granulocytic differentiation upon exposure to G-CSF, and shed light on the molecular mechanisms of maturation arrest in MDS cells.

DNA vaccines.

DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention and therapy of disease. Regarding the inoculation method of DNA vaccine, we recommend the gene gun delivery system, which is a highly reliable method compared to intramuscular inoculation. DNA vaccines could have potential advantages over other types of vaccines in that these vaccines can induce strong cellular immune responses, cytotoxic T lymphocytes and type 1 helper T cells, without resorting to live organisms or complicated protein formulation. The cellular immune responses are especially required for the protection against infections with intracellular pathogens such as viruses and Mycobacterium tuberculosis and protection against cancers, suggesting that they seem to be suitable targets of DNA vaccines. We describe here that their application to bacterial infections requires optimization of codon usage in the DNA vaccines to the host animal to improve translational efficiencies of the bacteria genes. DNA vaccines for a variety of pathogens and cancers have now entered phase I/II human clinical trials.

Expression of stanniocalcin in zona glomerulosa and medulla of normal human adrenal glands, and some adrenal tumors and cell lines.

Stanniocalcin (STC) is a calcium (Ca)-regulating hormone that was originally discovered in the fish Stannius body, which is a unique endocrine organ. Hypercalcemia increases STC secretion, which inhibits Ca uptake by the gills and normalizes serum Ca level. In this study we investigated the STC expression in human normal and abnormal adrenal cells. Immunohistochemistry using monoclonal antibody against STC revealed specific staining in zona glomerulosa and medulla of normal human adrenal glands. STC was also detected in human adrenal tumors, such as pheochromocytoma, differentiated neuroblastoma, and aldosterone-producing adenoma, and cultured adrenal tumor cells (rat pheochromocytoma PC-12 cells and human neuroblastoma NB-1 cells). However, undifferentiated human adrenal neuroblastoma was negative for STC staining. Reverse transcription polymerase chain reaction demonstrated STC mRNA expression in cultured PC-12 cells and NB-1 cells. Following several studies indicating that zona glomerulosa cells of adrenal glands express neuroendocrine properties, STC expression in normal and abnormal adrenal cells provides additional evidence to support the neuroendocrine differentiation of these cells. In conclusion, STC may be useful as a new cell marker of adrenal glands under physiological and pathological conditions.

Scapholunate dissociation caused by gouty arthritis of the wrist. Case report.

Gouty arthritis of the wrist is rare, and may be associated with scapholunate dissociation. To our knowledge, only two cases have been reported so far. In this report, we describe a 40-year-old patient with scapholunate dissociation caused by acute gouty arthritis of the wrist. His clinical findings and radiographs mimicked infectious arthritis or osteomyelitis of the carpal bones.

Radiosensitivity of human breast cancer cells transduced with wild-type p53 gene is influenced by the p53 status of parental cells.

Induction of apoptosis with chemotherapeutic agents or radiation in tumours is frequently related to the status of those p53 gene of the tumours. To examine whether forced expression of the wild-type p53 gene in tumour cells can modulate their susceptibility to radiation and anti-cancer agents, we retrovirally transduced two types of human breast cancer cell lines, which respectively harboured a mutated p53 gene (OCUB-M) or wild-type p53 gene (YMB-1), with the wild-type p53 gene. Transduced cells which consistently expressed the wild-type p53 gene (OCUB-M/p53, YMB-1/p53) proliferated at the same rate as control cells which were transduced with the beta-galactosidase gene (OCUB-M/lacz, YMB-1/lacz). However, sensitivity to radiation was increased in OCUB-M/p53 cells but not in YMB-1/p53 cells. In vitro chemosensitivity to DNA-damaging anticancer agents such as cyclophosphamide and 5-fluorouracil was not influenced by the transduction of the wild-type p53 gene in either cells. Expression of the wild-type p53 gene in p53-mutated human breast cancer cells can therefore increase their sensitivity to radiation but not their chemosensitivity. Therapeutic effects following by the transduction of the wild-type p53 gene were not observed in breast cancer cells already bearing the wild-type p53 gene.