The SF3B1 inhibitor spliceostatin A (SSA) elicits apoptosis in chronic lymphocytic leukaemia cells through downregulation of Mcl-1.

The pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukaemia (CLL), with high expression correlated with progressive disease. The spliceosome inhibitor spliceostatin A (SSA) is known to regulate Mcl-1 and so here we assessed the ability of SSA to elicit apoptosis in CLL. SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose- and time-dependent manner, but independently of SF3B1 mutational status, IGHV status and CD38 or ZAP70 expression. However, normal B and T cells were less sensitive than CLL cells (P=0.006 and P<0.001, respectively). SSA altered the splicing of anti-apoptotic MCL-1(L) to MCL-1(s) in CLL cells coincident with induction of apoptosis. Overexpression studies in Ramos cells suggested that Mcl-1 was important for SSA-induced killing since its expression inversely correlated with apoptosis (P=0.001). IL4 and CD40L, present in patient lymph nodes, are known to protect tumour cells from apoptosis and significantly inhibited SSA, ABT-263 and ABT-199 induced killing following administration to CLL cells (P=0.008). However, by combining SSA with the Bcl-2/Bcl-x(L) antagonists ABT-263 or ABT-199, we were able to overcome this pro-survival effect. We conclude that SSA combined with Bcl-2/Bcl-x(L) antagonists may have therapeutic utility for CLL.

Drug delivery observation of hydrophobe ferrofluid and magnetite nanoparticals by SPring-8 synchrotron radiation.

In this study, the composite magnetic nanoparticles of coated SiO nano film with about 8 nm size and high saturation magnetization value, were synthesized by liquid phase precipitation method. The magnetic nanoparticles can be dispersed in various liquid media, widely known as magnetic fluids or ferrofluids with both magnetic and liquid properties. The materials been collected great interests and more and more attentions to focus into Drug Delivery System (DDS) as a new technology in this paper. We use the composite nanoparticles to disperse H2O and inject the solutions into rat's in-vivo organs. And, in the experiments by using a strong photon beam of SPring-8 Synchrotron Radiation facility, the distribution stat and the effects of magnetic field as well as drug delivery behaviour of nanoparticles in the rat' kidney are verified by the in-vivo observations.

Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography.

Our previous studies suggest that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer. This study was conducted to determine whether alteration of RUNX3 gene expression could be detected in the normal-looking gastric remnant mucosa, and to ascertain any difference in the potential of gastric carcinogenesis between the anastomotic site and other areas in the remnant stomach after distal gastrectomy for peptic ulcer (RB group) or gastric cancer (RM group), by analysing RUNX3 expression with special reference to topography. A total of 89 patients underwent distal gastrectomy for gastric cancer from the intact stomach (GCI group) and 58 patients underwent resection of the remnant stomach for gastric cancer (RB group: 34 cases, RM group: 24 cases). We detected RUNX3 and gene promoter methylation by in situ hybridisation, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and methylation-specific PCR. The interval between the initial surgery and surgery for remnant gastric cancer (interval time) was 10.4 years in the RM group, and 27.5 years in the RB group. Cancers in the RB group were significantly more predominant in the anastomosis area (P<0.05). Within the tumour, downregulation of RUNX3 expression ranged from 74.7 to 85.7% in the three groups. The rate of downregulation of RUNX3 of adjacent mucosa was 39.2% (11 in 28 cases) in RB and 47.6% (10 in 21 cases) in RM, which are significantly higher than that of the GCI group (19.5%, 17 in 87 cases). In noncancerous mucosa of the remnant stomach in the RB group, RUNX3 expression decreased more near the anastomosis area. In the RM group, however, there were no significant differences in RUNX3 expression by sampling location. Based on RUNX3 downregulation and clinical features, residual stomach mucosa of the RM group would have a higher potential of gastric carcinogenesis compared to the RB or GCI group. Gastric stump mucosa of the RB group has higher potential especially than other areas of residual stomach mucosa. Measurement of RUNX3 expression and detection of RUNX3 methylation in remnant gastric mucosa may estimate the forward risk of carcinogenesis in the remnant stomach.

[Evaluation of spinal cord ischemia in endovascular stent graft repair and surgical operation of descending thoracic or thoracoabdominal aortic aneurysms].

Between October 1996 and June 2003, endovascular stent graft repair was performed in 87 patients with descending thoracic aortic aneurysms, graft replacement was performed in 24 patients with thoracoabdominal aortic aneurysms, and endovascular stent graft repair with concomitant surgical bypass of abdominal visceral arteries was performed in 3 patients with thoracoabdominal aortic aneurysms. The retrievable stent graft was inserted and evoked spinal cord potential were monitored in order to predict spinal cord ischemia for stent graft repair. There was no paraplegia or hospital death, although 3 patients had paraparesis in stent graft repair. Two of the 3 patients with paraparesis made a full neurologic recovery. There were no cases of paraplegia or paraparesis in surgical operations with thoracoabdominal aortic aneurysm. The concomitant surgical procedure was a good technique for patients in whom cardiopulmonary bypass could not be used. Our results of stent graft repair and surgical operation for descending thoracic or thoracoabdominal aortic aneurysms were acceptable. The retrievable stent graft was useful for prediction of spinal cord ischemia before endovascular stent graft repair of descending thoracic or thoracoabdominal aortic aneurysm.

A synthetic library of cell-permeable molecules.

Small molecules that induce or stabilize the association of macromolecules have proven to be useful effectors of a wide variety of biological processes. To date, all examples of such chemical inducers of dimerization have involved known ligands to well-characterized proteins. The generality of this approach could be broadened by enabling the discovery of heterodimerizers that target known macromolecules having no established ligand, or heterodimerizers that produce a novel biologic response in screens having no predetermined macromolecular target. Toward this end, we report the construction of a diversified library of synthetic heterodimerizers consisting of an invariant ligand that targets the FK506-binding protein (AP1867) attached to 320 substituted tetrahydrooxazepines (THOXs). The THOX components were generated by a combination of liquid- and solid-phase procedures employing sequential Mitsonobu displacements to join two structurally diversified olefin-containing monomers, followed by ruthenium-mediated olefin metathesis to effect closure of the seven-membered ring. The 320 resin-bound THOX ligands were coupled in parallel to AP1867, and the products were released from the resin to yield candidate heterodimerizers in sufficient yield and purity to be used directly in biologic testing. A representative panel of 25 candidate heterodimerizers were tested for their ability to pass through the membrane of human fibrosarcoma cells, and all were found to possess activity in this tissue culture system. These studies pave the way for further studies aimed at using small-molecule inducers of heterodimerization to effect novel biological responses in intact cells.

An improved rapid procedure for fluorescence in situ hybridization that is applicable to intraoperative cancer cytodiagnosis.

Fluorescence in situ hybridization (FISH) is among the most simple and useful methods for detecting numerical and structural aberration of chromosomes but it requires 12-24 h to complete. We devised a rapid FISH method that can be performed within 2 h. Here we describe the technique, which we have found to be extreme simple and as sensitive and specific as standard FISH, making it highly suitable for clinical use.

Genomic alterations in primary gastric cancers analyzed by comparative genomic hybridization and clinicopathological factors.

BACKGROUND/AIMS: Genetic changes during the oncogenesis and progression of gastric cancer remain unclear. The aim of our study was to analyze chromosomal aberrations in primary gastric cancers. METHODOLOGY: Using comparative genomic hybridization, we screened 47 primary gastric cancers for changes in the number of copies of DNA sequences. RESULTS: Gains of chromosome arms 20q (55%), 20p (36%), 17q (32%), 19q (30%) and 16p (30%), and losses of chromosome arms 4q (40%), 17p (40%), 5q (38%), 18q (30%) and 4p (28%) were detected most frequently. In addition, a high level of amplification was observed at 3q21 (2%), 6p21 (4%), 7q31 (6%), 8q23-24 (2%), 19q12-13 (2%), and 20q13 (2%). Among these alterations, the gain of 20q was the most frequent change. We then compared these changes with clinicopathological factors and identified signet ring cell carcinomas in 6 cases. Our study demonstrated no amplification of chromosome 20q in signet ring cell carcinoma in contrast to that in the other histologic types of gastric cancer. CONCLUSIONS: Our findings may be related to the morphologic and clinical features of signet ring cell carcinoma, and several oncogenes mapped on 20q may play an important role as determinants of the clinical and histologic features of gastric cancer.

[Hypertensive putaminal hemorrhage with extensive subarachnoid hemorrhage presenting extravasation of contrast material during angiography: case report].

The authors describe a case of hypertensive putaminal hemorrhage with extensive subarachnoid hemorrhage. On admission, the patient aged 71 presented right-sided motor weakness. CT scan on admission revealed left putaminal hemorrhage with extension into the ipsilateral thalamus and lateral ventricle as well as into the subarachnoid space of the suprasellar, ambient, interhemispheric and contralateral sylvian cisterns. To exclude vascular lesions, left carotid angiography was performed just after admission. The lateral view was unremarkable, but the anterior-posterior view demonstrated extravasation of contrast material from the left lateral lenticulostriate artery. The angiographic sylvian point was shifted to the lateral side. No abnormal vessels were revealed. CT scan after angiography showed exacerbation of both intracerebral and subarachnoid hemorrhages, but the consciousness level was unchanged. CT-guided stereotactic aspiration of the hematoma was performed 4 days after the onset, but failed to remove much hemtoma. The patient died of aspiration pneumonia 9 days after onset. The authors emphasize that extensive subarachnoid hemorrhage in cases with hypertensive putaminal hemorrhage may be an important finding which indicates high risk of rebleeding.

Inhibition of protein kinases by balanol: specificity within the serine/threonine protein kinase subfamily.

Balanol is a potent inhibitor of cyclic AMP-dependent protein kinase and protein kinase C, acting competitively with ATP with an affinity 3000 times that of ATP. We tested the capacity of balanol to inhibit representative serine- and threonine-specific protein kinases from the protein kinase subfamily that shares a common conserved catalytic core with cyclic AMP-dependent protein kinase. Balanol's pattern of interactions indicates considerable diversity of the ATP/balanol-binding sites of protein kinases within familial groups and even among isoforms of the same kinase. We propose that balanol is a protean structure that may be modified to produce selective, high-affinity inhibitors and probes of the ATP-binding sites of serine/threonine protein kinases.

Crystal structure of the potent natural product inhibitor balanol in complex with the catalytic subunit of cAMP-dependent protein kinase.

Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar Ki values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 A resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.

Knot formation in a long tube used in the treatment of a post-operative adhesive small bowel obstruction.

The complications specific to the management of the nasointestinal long tubes for small bowel obstruction are not generally appreciated. We report here of a case of knot formation of the long tube which was inserted for a 60 year-old male. Because it was difficult to place the tube in the distal stomach despite frequent changes in patient position, two coiled loops were noted at the fornix as the tip of the tube reached the antrum. The tube was gently removed without injury to the esophagus or stomach under fluoroscopic control. This case illustrates that when a long tube is used, the formation of multiple coils in the stomach should be avoided during placement to prevent knotting. Furthermore, a knotted tube can be gently removed non-operatively without esophageal injury. A short tube may be superior to a long tube because most of the complications can be avoided.

Analysis of histological therapeutic effect, apoptosis rate and p53 status after combined treatment with radiation, hyperthermia and 5-fluorouracil suppositories for advanced rectal cancers.

The tumour-suppressor gene p53 encodes a transcription factor that plays a critical role in the induction of G1 cell cycle arrest and apoptosis after DNA damage. To clarify the role of the p53 gene and apoptosis in combined hyperthermia, chemotherapy and radiation (hyperthermochemoradiotherapy, HCR therapy) for rectal cancer, we examined the histological response, rate of apoptosis, DNA fragmentation and p53 status in tumours from 28 patients undergoing HCR therapy before surgery and from 22 patients who did not have preoperative treatment. The therapeutic effect of HCR therapy was closely correlated with the rate of apoptosis; the correlation was statistically significant, suggesting that this effect occurs through apoptosis. The incidence of p53 mutations in the treated group were as follows: in tumours resistant to HCR therapy, four of seven (57.1%); intermediately sensitive, 7 of 13 (53.9%); or sensitive, three of eight (37.5%), suggesting that the therapeutic effect and apoptosis rate were related to the p53 status of the tumours to some extent, but the relation was not statistically significant. In the 22 control tumours (non-treated group), the apoptosis rate was 2.0 +/- 1.1%, and there was no significant difference in p53 status compared with the HCR group. Our study indicates that the pathological response to HCR therapy correlates with the rate of apoptosis with statistical significance and that it induces the therapeutic effect more significantly in rectal cancer cells with wild-type p53, although HCR therapy-induced apoptosis also occurs in some rectal cancers with mutated p53. Therefore, this combination therapy can induce an additive or synergistic anti-tumour effect in rectal cancers with wild-type p53 as well as in those with mutated p53 through apoptosis, offering new therapeutic opportunities and a better prognosis.

Interleukin-18 induces activation and association of p56(lck) and MAPK in a murine TH1 clone.

Interleukin-18 (IL-18) was identified as an inducer of interferon-gamma (IFN-gamma) production by stimulated T cells. In this study, we used an ovalbumin-responsive murine Th1 clone (OVA#4), in which DNA synthesis was reportedly enhanced after IL-18 treatment in the presence of a non-mitogenic TCR/CD3 stimulus, to examine signal transduction pathways. In the presence of the stimulus, IL-18 induced the appearance of tyrosine-phosphorylated proteins and herbimycin A inhibited DNA synthesis. It is suggested that protein tyrosine kinase (PTK) mediated signaling is induced by IL-18. Specifically, IL-18 induced phosphorylation of phosphorylates p56(lck) (LCK) and mitogen-activated protein kinase (MAPK). IL-18 alone induced the kinase activities of both LCK and MAPK, and the activities were increased by the TCR/CD3 stimulus. Simultaneously, IL-18 induced the association of LCK with MAPK and this was also increased by the TCR/CD3 stimulus. The activation of the LCK-MAPK pathway correlated with enhanced DNA synthesis in OVA#4 cells. These results suggest that the LCK-MAPK pathway is involved in IL-18 signaling and that IL-18 may play an important role in modification of TCR/CD3-mediated response.

Retinal light damage: protective effect of alpha-tocopherol.

We studied histologically the protective effect of alpha-tocopherol to retinal light damage. After 3-week-old albino rats were fed with an alpha-tocopherol deficient or supplemented diet and kept in a 12-hour dim light/12-hour dark environment for 8 weeks, each animal was exposed to intense light (2500 lux) for 1, 3, 6, 12, 24, and 72 hours. The eyes were enucleated and prepared for transmission electron microscopy study and image analysis of phagosomes. Before light exposure, the alpha-tocopherol content of the neural retina of the deficient and supplemented groups was 0.3 microgram and 23.34 micrograms, respectively. After 1- and 3-hour exposures, morphological changes in the retinal pigment epithelium and photoreceptor membranes were more extensive in the deficient group than in the supplemented group. After a 24-hour exposure, pyknotic photoreceptor nuclei were more numerous in the deficient group than the supplemented group. After 3-, 6-, and 12-hour exposures, large phagosomes were more numerous in the deficient group than in the supplemented group. These findings suggest that alpha-tocopherol can protect the retina from light injury for up to 24 hours of exposure.

Increased apoptosis rate by hyperthermochemoradiotherapy for advanced rectal cancers.

Apoptosis induced in cancer cells by ionizing radiation, hyperthermia, and 5-fluorouracil (5-FU), termed "hyperthermochemoradiotherapy" (HCR), has been well studied in vitro; however, the role of apoptosis in the tumocidal effect of HCR for primary rectal cancers has not yet been clarified. Therefore, we examined the relationship between the therapeutic effect and induction rate of histological apoptosis in 16 patients with rectal cancers after HCR. Numerous Tunel-positive apoptotic cells were found in the tumor tissue after HCR, but few were found in the tumors which had not received HCR. The histological therapeutic effect was closely correlated to the rate of apoptosis. Thus, we suggest that HCR induces a therapeutic effect mainly through apoptosis in human rectal cancers.

[The risk of palliative operation for bone metastasis].

Palliative operations for various bone metastases are being performed more frequently than before with the improvement of surgical technique and the development of new instruments. However, anesthesia in advanced cancer patients who are under palliative care accompanies inherent risks which are uncommon in general orthopaedic populations. Thus, we made a retrospective analysis of the medical records of 59 patients scheduled for palliative operation against metastatic bone lesions during the last 4 years. The survey revealed 5 cases of perioperative lethal events. Two of them died preoperatively from the hepatic failure and unexpected cerebral tumor embolism. An intraoperative cardiac arrest secondary to pulmonary embolism occurred in a patient during intramedullary nailing. There was a patient who developed disseminated intravascular coagulation and acute renal failure after posterior fixation of the lumbar vertebrae. The remaining one patient with superior vena cava syndrome developed life-threatening airway obstruction during general anesthesia with endotracheal intubation. We conclude that the exact evaluation of the patient's condition and the careful management of vital organ functions are mandatory during the perioperative period. In this regard, the anesthesiologists should be involved not only in the intraoperative anesthetic management but also in the perioperative care of the patients with bone metastasis.

Sporothrix schenckii isolated from a cat in Japan.

A yeast-like fungus isolated from a granulomatous and ulcerative skin lesion in a mongrel cat was mycologically examined. The isolate was identified as Sporothrix schenckii from the morphological findings and its pathogenicity in a mouse, confirming the first case of feline sporotrichosis in Japan. Fortunately, no transmission to humans occurred in this case, however the risk of humans contracting Sporothrix schenckii infection increases with the increase in the number of animals with sporotrichosis. Consequently when handling such animals protective clothing such as gloves should be worn to reduce the risk of transmission of the fungus through open wounds.

Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol.

BACKGROUND: The protein kinase C (PKC) family of serine/threonine-specific protein kinases is involved in many cellular processes, and the unregulated activation of PKC has been implicated in carcinogenesis. PKC inhibitors thus have significant potential as chemotherapeutic agents. Recently, the fungal metabolite balanol was shown to be an exceptionally potent inhibitor of PKC. We previously developed a practical and efficient total synthesis of balanol. We set out to use this synthetic molecule, and several synthetic analogs, to probe the mechanism of PKC inhibition and to determine the effect of balanol on the activity of other protein kinases. RESULTS: As well as inhibiting PKC, balanol is a potent inhibitor of cyclic AMP-dependent protein kinase (PKA), another protein serine/threonine kinase. Balanol does not, however, inhibit the Src or epidermal growth factor receptor protein tyrosine kinases. The inhibition of both PKC and PKA by balanol can be overcome by high concentrations of ATP, and molecular modeling studies suggest that balanol may function as an ATP structural analog. Although balanol discriminates rather poorly between PKC and PKA, only minor modifications to its molecular structure are required to furnish compounds that are highly specific inhibitors of PKA. CONCLUSIONS: A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.