Apatite containing aspartic acid for selective protein loading.

Physico-chemical modifications of hydroxyapatite (HAp) materials are considered as pre-requisites for the development of new bioactive carrier materials for drug delivery and tissue engineering applications. Since acidic amino acids have well-documented affinities to both HAp and basic proteins, HAp modified by aspartic acid (Asp, acidic amino acid) might be one of the candidate substrates for a basic protein carrier. Here, we synthesized HAp in the presence of various concentrations of Asp and observed that HAp crystallinity and other physico-chemical properties were effectively modulated. Detailed studies indicated that Asp was not incorporated in the HAp crystal lattice, but rather was trapped in HAp crystals. Protein adsorption studies indicated that the HAp particles modified by Asp had a selective loading capacity for basic protein. Therefore, HAp particles containing Asp might have potential in drug delivery applications, especially as the carrier of basic proteins including bFGF and BMP.

Effect of long term-administration with Gymnema sylvestre R. BR on plasma and liver lipid in rats.

Extract of Gymnema sylvestre leaves was administered to rats receiving either a high fat diet or normal fat diet for 10 weeks to investigate its influence on plasma and liver lipids and on visceral fat accumulation. In addition, its effect was compared with those of chitosan and the influence of combined use of these two substances was also evaluated. Within the high fat diet groups, the extract suppressed body weight gain and accumulation of liver lipids to the same extent as chitosan and the combined use. In addition, intraperitoneal fat and fat drop vacuoles on the epithelium of renal tubules, noted in the high fat diet group, were scattered by administration of the extract with the same results as for chitosan and combined use. Within the normal fat diet groups, plasma triglyceride levels decreased by administration of the extract, with similar results as chitosan and combined use. Concerning plasma total cholesterol, there was no decreasing effects with the extract, as found with chitosan and combined use. However, the effect of chitosan on plasma total cholesterol tended to be enhanced when used in combination with the extract. In addition, long-term administration of the extract did not show any influence on hematological and blood chemical parameters.

Effect of administration with the extract of Gymnema sylvestre R. Br leaves on lipid metabolism in rats.

Extract of Gymnema sylvestre R. Br leaves (GE) was orally administered once a day to rats fed a high fat diet or normal fat diet for 3 weeks to investigate its influence on lipid metabolism. As a result, GE did not influence body weight gain or feed intake in both diet groups during the experimental period. The apparent fat digestibility was significantly decreased by GE in both diet groups for the last 2 weeks of the experimental period, though not the apparent protein digestibility. In addition, the excretion of neutral sterols and acid steroids into feces was increased by GE in both diet groups. Furthermore, GE decreased the total cholesterol and triglyceride levels in serum. On the other hand, blood lecithin-cholesterol acyltransferase (LCAT) activity was increased by GE. Moreover, it was suggested that GE influenced cecal fermentation and that propionic acid and acetic acid contents in cecum were significantly increased by GE. Consequently, it was suggested that GE improved serum cholesterol and triglyceride levels through influence over a wide range of lipid metabolism in rats.

Neoadjuvant intra-arterial infusion chemotherapy combined with hormonal therapy for locally advanced breast cancer.

To determine the efficacy of combined neoadjuvant intra-arterial infusion chemotherapy and hormonal therapy for treating locally advanced breast cancer, we compared the outcomes of patients with or without this therapy, and also assessed histologic response. Ninety-four patients with locally advanced breast cancer (stage IIIa, 56; stage IIIb, 38). Nineteen stage IIIa and 17 stage IIIb patients received intra-arterial plus hormonal therapy while 37 stage IIIa and 21 stage IIIb patients with similar ages and follow-up durations did not. Treated patients received intra-arterial epirubicin plus oral medroxy-progesterone. Five-year disease-free survival rates were 77.5% for intra-arterially treated and 33.0% for other patients in stage IIIa, and 70.5% for intra-arterially treated and 38.1% for other patients in stage IIIb. Five-year overall survival rates were 94.4% for intra-arterially treated and 61.7% for other patients in stage IIIa, and 90.9% for intra-arterially treated and 56.3% for other patients in stage IIIb. Ten-year overall survival rates in stage IIIb were 90.9% for treated and 22.5% for other group patents. All differences were statistically significant (p<0.05). Good histologic response to intra-arterial therapy was seen in 75% of the primary tumors and 71% of involved lymph nodes. Neoadjuvant intra-arterial therapy with hormonal therapy yielded better survival rates than no intra-arterial therapy or our previous intra-arterial regimen.

Inhibitory effects of Sasa senanensis Rehder extract (SE) on calcium-ionophore A23187-induced histamine release from rat peritoneal exudate cells.

The effects of Sasa senanensis Rehder extract (SE) by alkaline hydrolysis on histamine release from rat peritoneal exudate cells (PECs) were examined. Preincubation with SE for 5 min suppressed calcium-ionophore A23187-induced histamine release in a concentration-dependent manner. A23187 evoked a quick increase in cytoplasmic free calcium ([Ca2+]i) levels in the presence of extracellular calcium. Preincubation with SE also had an inhibitory effect on calcium influx increases induced by A23187. These results indicate that SE prevents degranulation from rat PECs by inhibiting [Ca2+]i level elevation.

[A case report of Vp3 hepatocellular carcinoma responding to multidisciplinary treatment].

We report a 60-year-old man who had hepatocellular carcinoma with tumor thrombus in the main portal vein and left portal branch (VP3HCC). He was treated with transarterial chemo-embolization, surgical resection and intra-arterial infusion chemotherapy. He is now surviving without any sign of recurrence for 12 months after the initial therapy, even though the prognosis of VP3HCC is poor. This is a case in which the effect of multidisciplinary treatment was indicated.

cDNA cloning of Na+, K(+)-ATPase alpha-subunit from embryos of the sea urchin, Hemicentrotus pulcherrimus.

Na+, K(+)-ATPase alpha-subunit cDNA of the sea urchin, Hemicentrotus pulcherrimus, was obtained by twice screening prism and gastrula lambda gt10 cDNA libraries using an oligonucleotide probe derived from a mostly conserved region, FSBA (5'-p-(fluorosulfonyl)-benzoyladenosine) binding site of cation transport ATPases. The 5'-end of the non-coding region was determined by primer extension and the region was amplified by 5'-RACE method. The sea urchin alpha-subunit cDNA consists of 4401 nucleotides and encodes 1038 amino acid residues (MW, 114 kDa). The predicted primary structure, except N-terminal region, has similar degree of high homology to various metazoan Na+, K(+)-ATPase alpha-subunits. Alignment of amino acid sequence and a hydropathy profile also predicts eight putative transmembrane segments at least. The phylogenetic tree suspected from alignment of amino acid sequences of 21 species suggests that sea urchin and vertebrate Na+, K(+)-ATPase alpha-subunits seem to have evolved from a common origin, before vertebrate alpha-subunit divided into three isoforms.

Enhancement of cytokine production by macrophages stimulated with (1-->3)-beta-D-glucan, grifolan (GRN), isolated from Grifola frondosa.

The ability of grifolan (GRN), a purified fungal (1-->3)-beta-D-glucan, to induce various cytokines from macrophages was examined in vitro. Interleukin-6 (IL-6) activity in supernatants from the culture of macrophage cell line, RAW264.7 was dependent on increasing doses of GRN. The level of IL-6 induced with 500 micrograms/ml of GRN was comparable to that induced with lipopolysaccharide (LPS) 10 micrograms/ml. Enhancement of the mRNA level of IL-6 by treatment with GRN was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of GRN on production of IL-6 was also observed using peritoneal macrophages from C3H/HeJ mice which did not respond to endotoxins. This data suggested that the ability of GRN to activate IL-6 production of macrophages is not due to contamination of endotoxins in the preparation. Enhanced production of cytokine by GRN was observed not only with IL-6, but also with interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). In the production of TNF alpha, GRN was more effective than LPS used in this study. Other soluble or gel-forming(1-->3)-beta-D-glucans from various sources did not enhance the production of such cytokines although they are structurally similar to GRN. The above results indicate that GRN is a novel macrophage activator which augments cytokine production without dependence on endotoxins.

[The significance of intra-arterial infusion therapy for neoadjuvant chemotherapy].

Arterial infusion therapy with anticancer drugs is now attracting attention as a valuable modality for locally advanced breast cancer. Since 1977, we have used this therapy in 122 patients with primary breast cancer. The present report mainly discusses the clinical and histological response as well as the prognosis. The anticancer drugs were mainly given by two routes, infusion into the internal mammary artery and the subclavian artery. Continuous infusion of 5-FU and intermittent injections of MMC, ADR, 4'-epi-ADR and THP-ADR were jointly or individually made in each artery. Clinical response, defined as CR + PR, was noted in 48.4% of 5-FU group and 72.7% of ADR-MMC group. Histological response according to Shimosato Criteria, defined as grade IIb or better, appeared in 45.2% of main tumors and 25.4% of metastatic lymph nodes in the 5-FU group, and 70.9% main tumors and 46.3% of metastatic lymph nodes in the ADR-MMC group. The non-infusion group contained 27.7% of stage IIIb, against 72.2% in the infusion group. The 5-year overall survival rates were non-infusion group 62%, 5-FU group 34.1% and ADR-MMC group 66.2%. A significant difference was seen between the 5-FU infusion group and the ADR-MMC group (p = 0.03). Administration of high dose medroxy progesterone acetate for two weeks and 4'-epi-ADR infusion chemotherapy resulted in an excellent histological response. This combination therapy is a promising neoadjuvant chemo-endocrine therapy for advanced breast cancer.

Hepatocellular carcinoma after transcatheter hepatic arterial embolization. A histopathologic study of 84 resected cases.

BACKGROUND: Recently, transcatheter arterial embolization (TAE) has been used to treat hepatocellular carcinoma (HCC), yet much is still unknown regarding its optimal use. METHODS: Eighty-four patients with HCC after TAE underwent surgical resection. Fifty of the tumors were less than 3 cm (small HCC [S-HCC]), and 34 were 3 cm or larger (large HCC [L-HCC]). Necrosis rate, distribution of residual HCC, histopathology of the main tumor, and proliferating activity of residual HCC by means of proliferative cell nuclear antigen (PCNA) were examined. Twenty-two randomly selected patients with HCC treated with standard chemotherapy were used as non-TAE control subjects. RESULTS: A necrosis rate of greater than 95% was seen in 35 cases of S-HCC and in 15 of L-HCC. All five nonencapsulated tumors were L-HCC and had a much lower necrosis rate. No tumors in the control group showed a necrosis rate of greater than 95%. Encapsulated tumors were categorized according to their tumor interiors, capsules, and extracapsular zones. Complete necrosis of the tumor interior was 80.0% and 35.3% in S-HCC and L-HCC, respectively. Viable residual tumors were found mainly in the extracapsular zone in S-HCC, whereas in L-HCC they were located primarily in the tumor interior. Most capsules were affected by tumor necrosis and the subsequent healing process, resulting in a thick secondary capsule. Tumor interior necrosis was uniform and coagulative in S-HCC, in contrast to L-HCC, in which necrotic regions comprised several necrosis units of differing texture and were divided by fibrous septa. In contrast, the control group revealed spotty, sparse necrosis. Non-TAE tumor capsules were thin and pathologically characteristic of those naturally occurring in tumors, as opposed to the thick fibrous capsules, which are inducible by TAE therapy. In the TAE group, the PCNA positivity rates were 37.5%, 52.5%, and 100% in Grades 1, 2, and 3/4 combined, respectively. At the tumor-nontumor boundary of the extracapsular region, PCNA-positive cells were detected in 55.0% of the cases. CONCLUSIONS: The thickened tumor capsule serves as a good postoperative indicator of TAE response. Small tumors seem to be affected in the tumor interior, whereas extracapsular invasion undermines the TAE effect. PCNA was helpful in detecting the tumor-nontumor boundary and useful as a parameter of viability of HCC after TAE.

Effect of KCA-098, a new benzofuroquinoline derivative, on bone mineral metabolism.

The effect of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoli ne-6-one designated as KCA-098) on the bone mineral metabolism of chick embryonic bone was examined. KCA-098 dose-dependently inhibited bone resorption of cultured chick embryonic femora and calvariae. It increased the length, dry weight, and calcium and phosphorus contents of 9-d-old chick embryonic femurs cultivated for 6 d, indicating that it stimulated bone formation. These results show that KCA-098 has the unique effects of inhibiting bone resorption and stimulating bone formation of chick embryo. In addition, in an in vivo experiment, oral administration of KCA-098 (3.0 mg/kg/d) for 16 weeks led to an increase in calcium and phosphorus content as well as an increase in the amount of force required to break the femur from ovariectomized rats, suggesting that it may be useful for the treatment of bone diseases.

Chemoembolotherapy for recurrent hepatocellular carcinoma in the residual liver after hepatectomy.

The efficacy of transcatheter arterial chemoembolization using Lipiodol (TACE) to treat recurrent hepatocellular carcinoma (r-HCC) in the residual liver after radical hepatic resection was evaluated. During the last 8 years, TACE was performed in 68 patients with r-HCC for an aggregate total of 150 times. Of the 68 patients, 4 had a massive type r-HCC with tumor thrombus in the main portal vein (PVTT) at the time of the first TACE. Among the remaining 64 patients without PVTT, multiple r-HCCs were revealed in 46, and a single r-HCC in 18 by angiography and/or follow-up CT scans after the initial TACE. In 26 of the 68 patients (38.2%), at least one or more r-HCCs were fed not only by the hepatic arteries, but also by the extrahepatic collateral arteries, such as branches of the right inferior phrenic artery. The cumulative survival rates of these patients after hepatectomy and after the initial TACE for r-HCC were 98.6% and 87.1% for one year, 89.7% and 62.9% for 2 years, 74.0% and 34.3% for 3 years, 53.1% and 20.0% for 4 years and 40.3% and 0% for 5 years (mean survival duration: 1,647 days and 947 days), respectively. These results indicate that repeat TACE against r-HCC can help obtain long-term survival in patients with r-HCC. However, during TACE, we must give consideration to the newly developed collateral feeding artery to the r-HCC.

Clinical effects of complex spa therapy on patients with steroid-dependent intractable asthma (SDIA).

The clinical effects of spa therapy (swimming training in a hot spring pool+inhalation of iodine salt solution+fango therapy) were examined in 52 patients with steroid-dependent intractable asthma (SDIA). Clinical effects were found in 36 of the 52 (69.2%) patients with SDIA. The efficacy was higher in the patients between the ages of 41 and 50 (87.5%) and between 51 and 60 (84.2%) than in those in the other age groups. Regarding clinical asthma types, the efficacy of spa therapy was higher in patients with type Ia-2 (83.4%), type Ib (77.8%) and type II (80.0%) than in those with type Ia-1 (54.2%). Efficacy seemed to be related to airway inflammation: the proportion of neutrophils and eosinophils in bronchoalveolar lavage (BAL) fluid was higher in patients with marked and moderate improvement compared with those with slight and no improvement. Ventilatory function was improved by complex spa therapy. %MMF and %V25 values increased by more than 20% after therapy in patients with type Ia-2, Ib and II asthma. Bronchial hyperreactivity to methacholine was suppressed after therapy. Any decreased function of the adrenocortical glands expressed by low serum cortisol levels improved after therapy, accompanied by a reduction in the dose of glucocorticoids necessary to control asthma attacks.

Spa therapy improves ventilatory function in the small airways of patients with steroid-dependent intractable asthma (SDIA).

The improvement of ventilatory function by spa therapy was examined in 37 patients with steroid-dependent intractable asthma (SDIA) in relation to clinical asthma types. All subjects had been on long-term corticosteroid therapy before spa therapy. Spa therapy was found to improve the values of ventilatory parameters. The percent improvement in all subjects was +4.1% in %FVC, +8.9% in FEV1.0%, +9.8% in %PEFR, +22.0% in %MMF, +19.7% in %V50 and +28.2% in %V25, respectively. Relating to clinical asthma types, moderate improvement in %MMF and %V25 from 16.7% to 16.9% was observed in type Ia cases, and marked increase in %MMF, %V50 and %V25 was observed from 42.2% to 43.2% in type Ib cases. However, no significant increase was found in these parameters of types Ia or Ib after spa therapy. In patients with type II, a significant increase was shown in %V50 (p less than 0.05) and %V25 (p less than 0.01) after spa therapy. The results show that spa therapy improves the condition of small airways disorder in patients with SDIA.

[Effect, recurrence and prognosis of intra-arterial infusion chemotherapy with anticancer agents for advanced breast cancer--especially, the significance of tumor blood vessels].

Between January 1977 and March 1991, 86 patients of Stage III and Stage IV breast cancer were treated by intra-arterial infusion chemotherapy (IAIC) in Sapporo Medical College. The 5- and 10-year overall survival rates of Stage III b were: infusion group 53.1% and 53.1%, non infusion group 50.1% and 16.7%, respectively. A significant difference was seen in the 10-year survival rate (p less than 0.05). The 5- and 10-year overall survival rate of Stage III b histologically effective group was 67.3% and non-effective group 31.8%. A significant difference was seen between these two groups (p = 0.0241). A significant difference was seen between n2 positive Stage III b infusion group and non-infusion group in 7-year survival rate (p less than 0.01). The ischemic effect on tumor cells by degeneration changes of the blood vessels was important for the histological effect. Pre-operative angiography helped in the choice of anticancer agents. The sufficient adjuvant chemo-endocrine therapy will assure a good prognosis for IAIC-treated Stage III b breast cancer patients.

Effects of single and multiple moxibustions on activity of platelet function, blood coagulation and fibrinolysis in mice.

The effects of single and multiple moxibustions on platelet function, blood coagulation and fibrinolytic activity in ddY mice were studied. The increase in platelet aggregation and ATP-release after a single moxibustion was dependent on moxa weight and the kind of platelet stimulus. Blood coagulative activity tended to increase in the early phase after a single moxibustion. However, multiple moxibustions maintained the homeostasis on blood coagulation and fibrinolytic activity. This investigation suggests that the effects of moxibustion on platelet functions and coagulative and fibrinolytic activities cause an enhancement of the phagocytic activity in the host defense mechanism.

Effects of single moxibustion on cutaneous blood vessel and microvascular permeability in mice.

The effects of a single cone moxibustion on cutaneous blood vessel and microvascular permeability in ddY mice have been studied by microscopical observation and by the dye leakage technique. In our results, moxibustion induced some kind of inflammatory response such as the blood vessel reaction with leukocytes exudation and the enhancement of microvascular permeability, which is considered one of the host defense mechanisms.

Quantitative Determination of Vinblastine in Tissue Cultures of Catharanthus roseus by Radioimmunoassay.

A radioimmunoassay has been developed for the quantitative determination of small amounts of vinblastine in tissue cultures of CATHARANTHUS ROSEUS (L.) G. Don. Antibody was obtained by the immunization of rabbits against a conjugate of vinblastine with bovine serum albumin. The antibody had a high affinity (Ka = 1.2 x 10 (9)l/mol) and specificity for vinblastine. The usable range of standard curve for assay was 0.5-10 ng/ml. Crude alkaloid extracts of tissue cultures could be assayed and many samples could be processed in one time. It was found that the vinblastine contents of multiple shoot cultures were lower than that of intact plants but much higher than that of callus cultures.

[Effects of antipyretics and anticancer drug on human cells at high temperature condition].

The effects of antifebriles and anticancer drug on human vascular endothelial cells (HVE) and several cultured human cells were investigated. The HVE were isolated from umbilical cord veins by enzyme treatment and cultured successively in aerated synthetic medium, RPMI-1640, with 20% preclostrum new born calf serum. The presence of factor VIII antigen in the HVE was determined by enzyme-labeled antibody method. Cell count and protein amount were examined at regular intervals. At 3 hour-expose, sulpyrine was more toxic to the cultured cells than aspirin at 37 degrees C. The cytotoxicity of sulpyrine was markedly enhanced at 40 degrees C than at 37 degrees C. However, there was no enhancement in the cytotoxicity of aspirin at 40 degrees C. Cultured HVE and normal human fetal lung (HAIN-55) cells at 37 degrees C were sensitive to sulpyrine, and their sensitivity of the cells to the drug were markedly enhanced when they were incubated at 41 degrees C. In contrast, sensitivity of malignant human cells (HeLa cells) to sulpyrine was not found at 37 degrees C, however sensitivity of the cells to the drug was manifested at 41 degrees C of incubation. There was no effect of 5-fluorouracil (FU) on the growth of HVE and HAIN-55 cells at 41 degrees C, while HeLa cells showed high susceptibility to FU at the same temperature. The results showed the possibility that normal human cells may be sensitive to antifebrile drugs but not to anticancer drug at ordinary and high temperature, whereas malignant human cell may be susceptible to both antifebrile drugs and anticancer drug at high temperature.