Photoreceptor differentiation of retinoblastoma: An electron microscopic study of 29 retinoblastomas

Retinoblastomas exhibit a unique form of differentiation to produce cell elements similar to those seen in a photoreceptor cell. An ultrastructural study was performed on 29 cases of retinoblastoma to further clarify the cytologic characteristics of the tumor cells. The age of the retinoblastomas averaged 17.1 months and the tumor cells showing photo-receptor differentiation were demonstrated in 10 cases (35%). The findings were especially notable in retinoblastomas with Flexner-Wintersteiner rosette formation (seven cases, 28%). Similar photoreceptor differentiation was also evident in solid cell clusters without rosette formation (four cases, 14%). The presence of photoreceptor elements was assumed to be significantly frequent both in Flexner-Wintersteiner rosettes and in the solid cell clusters. The cell cytoplasm also showed proliferation of long mitochondria and microtubules, reflecting photoreceptor differentiation. The hereditary-type retinoblastoma showed more advanced cell differentiation than the non-hereditary type. Photoreceptor differentiated retinoblastoma showed rather indolent growth compared with the undifferentiated type, and the former can expect a curative treatment by operation. These observations provide additional findings of the biological nature of retinoblastomas.     

Immunohistochemical dynamics of leukotoxin (9.10-eooxv-12-octadecenoic acid) in lungs of rats

This paper investigates the immunohistochemical dynamics of leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx) in the lungs of rats exposed to hyperoxia with or without paraquat. The rats were treated with 100% oxygen or ambient air for 24. 48, 72 and 96 h in the presence or absence of a low or high dose paraquat (1,1-di-methyl-4,4-bipyridinium, PQ) injection. Immunostaining for LTx demonstrated positive reactions in the neutrophils that showed a progressive increase in intensity of staining with time in all groups exposed to 100% oxygen and in the group with high dose PQ, but the positive findings were weak in the group injected with low dose PQ only. We found the positive immunostaining reaction not only in neutrophils but also in alveolar macrophages. This indicates that LTx is produced by alveolar macrophages as well as by neutrophils depending on the treatment period under hyperoxic conditions, suggesting that LTx is an important chemical mediator in pulmonary diseases.     

Haematological improvement by long-term administration of recombinant human granulocyte-colony stimulating factor and recombinant human erythropoietin in a patient with severe aplastic anaemia

A 6-year-old girl with post-hepatitic severe aplastic anaemia was referred to our hospital. Haematological examination showed a haemoglobin level of 5.2 g/dl, platelet count of 8,000/l, and white blood cell count of 130/l with 17% neutrophils. She was treated with recombinant human granulocytecolony stimulating factor (15 g/kg/day i.v.) and cyclosporin A (6 mg/kg/day p.o.). The absolute neutrophil count gradually increased, but Hb and platelets were not improved. The intravenous administration of recombinant human erythropoietin (100 U/kg three times a week) was started, and the reticulocyte count reached 20000/l on day 12. The platelets increased to 81000/l after 16 months of combined administration of recombinant human granulocyte-colony stimulating factor, recombinant human erythropoietin and cyclosporin A. After 20 months of combined administration, the haematological results were: Hb, 13.1 g/dl; platelets 80000/l: WBC, 9500/l with 40% neutrophils. After recombinant human granulocyte-colony stimulating factor treatment, the myeloid elements of the bone marrow and the number of granulocyte-macrophage colony forming units increased. Bone marrow erythropoiesis and erythroid colonies also increased after recombinant human erythropoietin administration. The clinical course suggested a beneficial effect of haemopoietic growth factors and cyclosporin A in post-hepatitic aplastic anaemia.     

Autogenous onlay grafting for enhancement of extracortical tissue formation over porous-coated segmental replacement prostheses

BACKGROUND: Prosthetic reconstruction with extracortical bone-bridging is an effective method of limb salvage after resection of a malignant or locally invasive benign bone tumor. Use of cancellous bone graft alone is less effective in achieving extracortical bone-bridging. The present study was performed to investigate the effects of a corticocancellous onlay graft on bone and soft-tissue formation over a porous-coated replacement prosthesis in the mid-diaphyseal region of canine femora. METHODS: Bilateral resection of a six-centimeter segment of the femoral diaphysis and reconstruction with a porous-coated segmental prosthesis was performed in six mongrel dogs. In one limb (the experimental side), eight strips of corticocancellous bone were evenly placed around the junctions between the femur and the prosthetic surface. Cancellous bone was placed under and between the strips of cortical bone. No graft was used in the other limb (the control side). The animals were followed for twelve weeks, with sequential assessments of load-bearing and radiographic evaluation. Biomechanical, histological, and microradiographic analyses of the specimens were performed after death. RESULTS: On the control side, load-bearing at four weeks postoperatively was significantly decreased compared with the preoperative value (p<0.05); no difference in these values could be detected on the experimental side. Both the area of the callus and the contact area between the bone and the prosthetic shoulder were greater on the experimental side (p<0.05). The mechanical stiffness and the maximum torque at failure of the extracortical bridging tissue across the junction between the bone and the prosthetic shoulder were eighteen (p<0.007) and five times greater (p<0.05), respectively, on the experimental side. CONCLUSIONS: Extracortical bone-bridging was accomplished with corticocancellous onlay bone-grafting. Without bone-grafting, bone formed only occasionally. Bone-grafting also enhanced the formation of a soft-tissue capsule around the prosthesis.     

Caspase activation during apoptotic cell death induced by expanded polyglutamine in N2a cells

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder. To investigate the mechanism of neurodegeneration induced by mutant huntingtin, we developed a stable neuro2a cell line expressing truncated N-terminal huntingtin (tNhtt) with EGFP using the ecdysone-inducible system. The formation of aggregates and the cell death induced by expression of tNhtt with expanded polyglutamine was repeat length- and dose-dependent. Caspases were activated, and the death substrates of caspases, lamin B and ICAD (an inhibitor of caspase-activated DNase), were cleaved in this cell death process. The cleavage of lamin B was inhibited by caspase inhibitors. These findings suggest that the cell death induced by tNhtt with expanded polyglutamine is mediated by caspases.     

Ser203 as well as Ser204 and Ser207 in fifth transmembrane domain of the human beta2-adrenoceptor contributes to agonist binding and receptor activation.

We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.     

L-cis diltiazem attenuates intracellular Ca(2+) overload by metabolic inhibition in guinea pig myocytes

We have previously demonstrated that treatment with L-cis diltiazem reduced cardiac infarct size in vivo. To examine the effect of L-cis diltiazem on Ca(2+) overload induced by ischemia/reperfusion, we used a model for Ca(2+) overload produced by metabolic inhibition in isolated guinea pig myocytes. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was quantified by fura-2 fluorescence microscopy and Ca(2+) overload was induced by inclusion of 1 microM of carbonyl cyanide m-chrolophenylhydrazone (CCCP) for 40 min treatment followed by washout for 30 min. This treatment caused a large [Ca(2+)](i) elevation as well as a sustained contracture of the cardiomyocytes. The increase was suppressed by 10 microM of 2-[2-[4-(4-nitrobenzyloxy) phenyl] ethyl] isothiourea methanesulphonate (KB-R7943), a specific inhibitor of the Na(+)/Ca(2+) exchanger, but not by nitrendipine (10 microM). L-cis Diltiazem (10 microM) attenuated the [Ca(2+)](i) increase, suggesting that L-cis diltiazem elicits a cardioprotective effect via attenuation of the [Ca(2+)](i) increase induced by metabolic inhibition and energy repletion.     

Pharmacological evaluation of the development candidate

In recent years, drugs developed in Japan are used internationally in other countries. The pharmaceutical pre-clinical phase in critical in the drug developing cycle. Pharmacologists contribute to finding the leading compounds and evaluate the right compound for clinical trial. The selection of the right compound for development will save both time and money. In the pharmacological evaluation of a development candidate, in vivo animal studies that can assure the clinician that the drug is both effective and safe are usually required. Studies on the mechanism of action of the drug and how the drug works in the diseased state becomes important since a known target molecule usually serves as the basis for the development of a new drug.     

Treatment with l-cis diltiazem before reperfusion reduces infarct size in the ischemic rabbit heart in vivo.

l-cis Diltiazem, an optical isomer of diltiazem, protects against myocardial dysfunction in vitro, whereas its Ca2+ channel blocking activity is about 100 times less potent than that of diltiazem. However, there is no evidence that l-cis diltiazem actually protects against ischemia/reperfusion injury in vivo. To assess this, we employed an anesthetized rabbit model, where the left circumflex artery was occluded for 15 min and reperfused for 360 min. Treatment with diltiazem before and during ischemia (bolus 200 microg/kg and 15 microg/kg per minute for 25 min, i.v.; 575 microg/kg total) showed slightly depressed hemodynamic parameters, while l-cis diltiazem (1150 microg/kg) had no effect. Treatment with l-cis diltiazem produced a high recovery of the thickening fraction and limited the infarct size in a dose-dependent manner. Furthermore, the treatment with l-cis diltiazem (1150 microg/kg) or diltiazem (575 microg/kg) 5 min before reperfusion also limited the infarct size, but not after reperfusion. These results suggest that l-cis diltiazem affects some events in the onset of reperfusion, independently of Ca2+-channel-blocking action. Our observations are the first to show that l-cis diltiazem demonstrated its cardioprotective action in the ischemic rabbit heart in vivo.