Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany. In the present study, to clarify the pharmacological properties of vasodilation produced by GBE, we examined the effect of GBE and quercetin, one of the ingredients in GBE, on the thoracic aorta isolated from Wistar rats. GBE produced a dose-dependent relaxation in the aortic ring precontracted with noradrenaline, and the relaxation was abolished by L-N(G)-nitro arginine methyl ester (L-NAME). Quercetin produced a similar relaxation, which was also abolished by L-NAME. We then examined the effects of GBE and quercetin on the intracellular calcium level ([Ca2+]i) of cultured aortic endothelial cells using a fluorescent confocal microscopic imaging system. Both GBE and quercetin produced significant increases in [Ca2+]i in the endothelial cells. The increase in [Ca2+]i by quercetin (10(-6) M) was abolished by removing the extracellular Ca2+, but was not affected by thapsigargin, a calcium pump inhibitor. These findings suggest that a principal ingredient of GBE producing vasodilation is quercetin, which can activate nitric oxide synthesis and release by increasing [Ca2+]i in vascular endothelial cells.

Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer.

The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.

Multilineage cells from human adipose tissue: implications for cell-based therapies.

Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

Clinical application of biodegradable rib connecting pins in thoracotomy.

BACKGROUND: To assess the usefulness in rib reconstruction of biodegradable connecting pins made of polylactide (PLA), PLA rib pins were compared with conventional non-absorbent aluminous ceramic rib pins (alumina rib pins) in terms of their ability for fixing and healing of cut ribs in thoracic surgery. METHODS: There were 13 cases of rib fixation after thoracotomy using PLA rib pins and 11 cases using alumina rib pins, all of which were inserted into the medulla of the stump of the ribs that had been cut at thoracotomy to secure a larger surgical field. The observation period was 6 months after the operation. RESULTS: The degree of vertical shift of the ribs connected with PLA rib pins was significantly reduced compared with the degree using alumina rib pins. In chest radiographs clear zones around the pins, indicating a delay in bone neogenesis, were observed frequently around the alumina rib pins but were absent around the PLA rib pins. Osteosynthesis using PLA rib pins was significantly more favorable than with alumina rib pins. CONCLUSIONS: These results demonstrated that the PLA rib pin was superior to the alumina rib pin for fixing and healing of the cut rib.

[Thymidylate synthase expression in patients with hypopharyngeal carcinoma].

We analyzed the relationship between clinical response to neo-adjuvant chemotherapy including 5-fluorouracil (5-FU) in patients with hypopharyngeal carcinoma (HPC) and thymidylate synthase (TS) expression in their tumors. TS expression was evaluated with immunohistochemical staining techniques on biopsy specimens from HPC patients. TS immunostaining was divided into four levels (TS0-TS3) according to its level and pattern. The relationship between prognosis, tumor size, nodal status, differentiation of tumor cells and TS expression were also investigated. There was a statistically significant association between the level of TS expression and tumor size (p < 0.01). In terms of the effectiveness of chemotherapy, tumor differentiation, nodal status and prognosis, a statistical difference was not found in TS expression. These results suggest that the level of TS expression may show the degree of tumor proliferation, but may not necessarily be useful to obtain a response to chemotherapy including other drugs, e.g., cisplatin and other derivatives of platinum.

Indication of chemoembolization therapy without gelatin sponge for hepatocellular carcinoma.

This study evaluated the effect of chemoembolization (C-LIP) consisting of ethiodized oil (Lipiodol Ultra Fluid; André Guerbet, Aulnay-sous-Bois, France) and epirubicin, without gelatin sponge on hepatocellular carcinoma (HCC), administered by hepatic arterial infusion. We analyzed the cases from two points of view: the local recurrence rate for hypervascular solitary small HCC (tumor size: < or =3 cm in diameter) and the cumulative survival rate for advanced HCC (stage VI according to the criteria of Liver Cancer Group of Japan) following C-LIP therapy. The C-LIP also was compared with transcather arterial embolization (TAE; C-LIP followed by gelatin sponge) and percutaneous ethanol injection therapy (PEIT). In the small HCC cases, the recurrence rate at 1 year after C-LIP was 77% (10 of 13 patients), while the local recurrence rate was 46% (six of 13 patients) at 6 months and 61% (eight of 13 patients) at 1 year. The local recurrence rate at 1 year was 29% (four of 14 patients) after TAE and 20% (three of 15 patients) after PEIT. These results showed that the effect of local anticancer therapy by C-LIP was not as potent as that of TAE or PEIT. In advanced HCC cases, the cumulative survival rate for 13 patients treated by C-LIP was 72% at 6 months, 36% at 1 year, and 14% at 2 years. However, the survival rates for 13 patients at 6 months, 1 year, and 2 years after TAE were 46%, 23%, and 8%, respectively. There was no difference between the C-LIP patients and TAE patients with regard to the pretreatment liver function. Three patients died within 2 months after the initial TAE. These deaths were mainly due to damage to the noncancerous liver parenchyma. Therapy with C-LIP alone was not appropriate for hypervascular solitary small HCCs, and additional treatment was necessary. We think C-LIP therapy should be selected instead of TAE for advanced HCCs to avoid severe parenchymal damage.

[Primary cutaneous lymphoma--mycosis fungoides].

Malignant lymphoma of the skin is a type of extranodal lymphoma with a benign prognosis, in which the main organ involved is the skin. Some 80-90% of the cases in Japan show a T-cell phenotype. Mycosis fungoides and Sézary syndrome are common T-cell lymphomas of the skin. The tumor cells of mycosis fungoides, small and medium-sized cells with cerebriform nuclei, are detected in an epidermo-dermo junction. The tumor cells show CD3, CD4 and CLA, (cutaneous lymphocyte associated antigen) positivity. Various forms of topical therapy, such as topical steroid, photochemotherapy (PUVA), and interferons, have been indicated for the good-risk group (stages I A, I B and II A). Electron-beam irradiation, various chemotherapy, such as low-dose etoposide, low-dose MTX and CPT-11 and deoxy coformycin (DCF) plus IFNs, have been indicated for intermediate-risk group (stage II B, III and IV A). BRMs plus low-dose etoposide, electron-beam irradiation and a multiagent combination chemotherapy, such as MACOP-B, M-BACOD or ProMACE-CytaBOM, have been indicated for the high-risk group (stages IV A and IV B). Cutaneous B cell lymphoma (CBCL) can be diagnosed using a molecular biological assay. The tumor cells of CBCL do not express T-cell antigens such as CD2, CD3 and CD43 and show B-cell antigens such as sIg, CD19, CD20 and CD22. Electron-beam irradiation has been indicated for early-stage CBCL (stages I and II). An effective multiagent combination chemotherapy, such as MACOP-B, M-BACOD or ProMACE-CytaBOM, is required for patients with advanced stage CBCL (stages III and IV).

In vitro evaluation of linear motor-driven total artificial heart.

This paper deals with the in vitro evaluation of a newly developed linear motor-driven total artificial heart (linear TAH). The linear TAHs have been developed and evaluated in mock testing and acute animal experiments. The new linear TAH was made based on experience with acute animal experiments. The maximum static thrust of the new linear pulse motor is 146 N with exciting current of 1.69 A. The weight and the volume of this linear TAH are 1.9 kg and 560 ml, respectively. The linear TAH has a kinetic thrust of 85 to 43 N and provides the maximum flow rate of 6.8 L/min in mock circulatory testing.

Molecular cloning of a human transmembrane-type protein tyrosine phosphatase and its expression in gastrointestinal cancers.

To determine the expression of various protein-tyrosine phosphatases (PTPs) in human gastric cancers, cDNAs encoding conserved PTP domains were amplified by reverse transcriptase polymerase chain reaction from KATO-III cell mRNA and sequenced. Among 72 polymerase chain reaction clones, one of the cDNA sequences encoded a novel potential PTP (stomach cancer-associated PTP, SAP-1). The full length (3.9 kilobases) of the SAP-1 cDNA was further isolated from the KATO-III cell cDNA library and the WiDr cell cDNA library. The predicted amino acid sequence of the SAP-1 cDNA showed that mature SAP-1 consisted of 1093 amino acids and a transmembrane-type PTP, which possessed a single PTP-conserved domain in the cytoplasmic region. The extracellular region of SAP-1 consisted of eight fibronectin type III-like structure repeats and contained multiple N-glycosylation sites. These data suggest that SAP-1 is structurally similar to HPTP beta and that SAP-1 and HPTP beta represent a subfamily of transmembrane-type PTPs. SAP-1 was mainly expressed in brain and liver and at a lower level in heart and stomach as a 4.2-kilobase mRNA, but it was not detected in pancreas or colon. In contrast, among cancer cell lines tested, SAP-1 was highly expressed in pancreatic and colorectal cancer cells. The bacterially expressed SAP-1 fusion protein had tyrosine-specific phosphatase activity. Immunoblotting with anti-SAP-1 antibody showed that SAP-1 is a 200-kDa protein. In addition, transient transfection of SAP-1 cDNA to COS cells resulted in the predominant expression of a 200-kDa protein recognized by anti-SAP-1 antibody. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2.