Inhibitory effects of (-)-epigallocatechin-3-gallate from green tea on the growth of Babesia parasites.

(-)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and accounts for 50-80% of the total catechin in green tea. (-)-Epigallocatechin-3-gallate has antioxidant, anti-inflammatory, anti-microbial, anti-cancer, and anti-trypanocidal activities. This report describes the inhibitory effect of (-)-Epigallocatechin-3-gallate on the in vitro growth of bovine Babesia parasites and the in vivo growth of the mouse-adapted rodent babesia B. microti. The in vitro growth of the Babesia species was significantly (P<0.05) inhibited in the presence of micromolar concentrations of EGCG (IC50 values=18 and 25 microM for B. bovis, and B. bigemina, respectively). The parasites showed no re-growth at 25 microM for B. bovis and B. bigemina in the subsequent viability test. The drug significantly (P<0.05) inhibited the growth of B. microti at doses of 5 and 10 mg/kg body weight, and the parasites completely cleared on day 14 and 16 post-inoculation in the 5 and 10 mg/kg treated groups, respectively. These findings highlight the potentiality of (-)-Epigallocatechin-3-gallate as a chemotherapeutic drug for the treatment of babesiosis.

cDNA cloning of calcineurin heterosubunits from the pheromone gland of the silkmoth, Bombyx mori.

Pheromone biosynthesis activating neuropeptide (PBAN) stimulates the step of fatty acyl reduction in the pheromone biosynthetic pathway of the silkmoth, Bombyx mori. It has been suggested that the intracellular signal transduction of PBAN in B. mori involves Ca(2+), calmodulin, and calcineurin (also known as protein phosphatase 2B). We have cloned two cDNAs encoding calcineurin heterosubunits from a pheromone gland cDNA library of B. mori. The 2,996-bp clone predicts a 495-amino acid protein homologous to the catalytic subunit calcineurin A (CnA) with a molecular mass of 55,968. The deduced amino acid sequence well conserves the calcineurin B (CnB)-binding domain and two subdomains, a calmodulin-binding and an autoinhibitory domain, showing 77-85% and 82% identities to the isoforms of Drosophila melanogaster CnA and human CnA, respectively. On the other hand, the 820-bp clone predicts a 170-amino acid protein homologous to the regulatory subunit CnB with a molecular mass of 19,357. The deduced amino acid sequence well conserves four EF-hand type calcium-binding structures, showing 95% and about 85% identities to D. melanogaster CnB and mammalian CnBs, respectively. A yeast two-hybrid system has demonstrated the molecular interaction between B. mori CnA and CnB. Northern blot analyses revealed that both CnA and CnB genes were expressed in various larval and adult tissues of B. mori. Both transcripts detected in the pheromone gland three days before adult eclosion increased by the day before eclosion and the mRNA levels were found to be high even two days after adult eclosion. Immunohistochemical analysis has revealed that B. mori calcineurin is localized in the cytoplasm of the pheromone-producing cells.

[A case of breast cancer with multiple organ metastases responding remarkably to combination therapy of CAF (cyclophosphamide, adriamycin and 5-FU), 5'-DFUR and MPA (medroxyprogesterone acetate)].

A 52-year-old woman complaining of breast tumor was diagnosed as having advanced breast cancer (T4bN1M1-Stage IV), with metastasis of multiple organs (lung, liver, mediastinal and unilateral axillary lymph nodes) after which she underwent tumorectomy. Postoperative adjuvant therapy was performed using combined chemoendocrine therapy (CAF + 5'-DFUR + MPA). Following the endocrine therapy, the metastatic lesions of the liver and lung had disappeared. The adverse effects were not remarkable. Complete remission was continued for 2 years and 3 months, and the patient enjoyed a favorable quality of life.

Antitumor effect of S-1 on DMH induced colon cancer in rats.

The aim of this study was to establish an autochthonous colon cancer model in the rat as an in vivo secondary screen for the general evaluation of new anticancer agents against colorectal cancer, and also to evaluate practically the antitumor activity of 1M tegafur-0.4M 5- chloro-2,4-dihydroxypyridine-1M potassium oxonate(S-1), a new p.o. fluoropyrimidine. Thirty-two Sprague-Dawley rats received dimethlhydrazine(40 mg/kg) s.c. once weekly for 10 weeks to induce colon cancer.20 weeks after beginning the carcinogen treatment, a barium enema was performed to visualize tumors. The animals were divided into a control group and S-1 treatment group. After 5 weeks of treatment, the barium enema was repeated. The mean doubling time of 24 tumors in the control group was 19.0 + 8.4 (SD) days. Response to S-1 was judged as effective when the doubling time exceeded 35.8 days, calculated from the mean + 2SDs in the control group. The response rate of S-1 was 55%, 34% of the tumors were decreased in size after treatment. This figure was higher than that of clinically-used 5-fluorouracil(5-FU) derivatives; 5-FU;6%, Tegafur(FT):6%, 1M tegafur-4M uracil(UFT):14%, reported in our previous study. An autochthonous colon cancer model is useful to evaluate the clinical therapeutic efficacy of drugs for colorectal cancer, and S-1 is expected to have a high therapeutic effect on human colorectal cancer.

Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

[Histological effect of arterial embolization chemotherapy for metastatic liver tumors from colorectal cancer--report of cases of hepatectomy after arterial embolization chemotherapy].

To evaluate the histological effect of arterial embolization chemotherapy (AEC) for the metastatic liver tumors from colorectal cancer, 5 lesions were examined in 4 patients who were treated with surgery after AEC. In addition, to compare the histological changes of the metastatic liver tumors, we histologically examined 7 lesions in 5 patients who were treated with surgery alone as a historical control. For the patients with AEC, anti-cancer agents and lipiodol were administered by selected hepatic arterial infusion with fragments of sponzel. In a group of AEC, all metastatic liver tumors were detected after resection of the primary tumors. The range of tumor size in these lesions was from 2.5 to 4.2 cm in diameter. Otherwise, in a group of non-AEC, the size of tumors ranged from 2 to 5 cm in diameter. In angiography, tumor stainings were detected in 2 lesions, and in all lesions no accumulation of lipiodol was detected by CT scan. The overall response rate at the surgery was 7 to 33% (average 23%) in a group of AEC. In addition, in 2 lesions, about 60% of the lesions were necrotic tissues, and more than 90% of the tumor lesions in 3 lesions were histologically necrosis. Within these lesions, encapsulation of the metastatic tumors was observed in 3 lesions. Otherwise, in a group of non-AEC, 50-70% of the tumor lesions were histologically detected as necrosis, and in one lesion, encapsulation was observed. These evidences suggest the histological effect of AEC may be evaluated when more than 70% of tumor necrosis in lesions is observed. In conclusion, the treatment was effective in 3 cases and not effective in one case.

A case of adrenal insufficiency due to acquired hypothalamic CRH deficiency.

A 40-year-old woman with adrenal insufficiency was clinically diagnosed and examined with human corticotropin releasing hormone (CRH). This patient with secondary hypo-adrenalism has shown a normal serum cortisol response to exogenous ACTH administration and has been examined with CRH, lysine-vasopressin (LVP) and insulin tolerance test (ITT), respectively. Success in secreting ACTH in response to both CRH and LVP tests, but not ITT, suggests that this disorder was possibly due to a hypothalamic CRH deficiency rather than pituitary corticotroph dysfunction. A combination of the CRH test and ITT has come to play an increasingly significant role in the diagnosis and differential diagnosis of isolated ACTH deficiency syndrome.

[Local eosinophilia of the nose, the larynx and the trachea in rats sensitized with Japanese cedar pollen].

Eight Brown Norway rats were immunized twice at days 0 and 13 by intraperitneal injections of 10 micrograms Cry j I, one of major allergen to Japanese cedar pollinosis, mixed with 4.5 mg aluminium hydroxide gel. Serum level of Anti-Cry j I IgE antibody was detected by the method of ELISA. Mean value of serum levels of specific IgE to Cry j I in the sensitized rats was significantly higher than that in the non-sensitized five rats (p < 0.01). The grades of eosinophil and lymphocyte accumulation in the nasal mucosa of the sensitized rats were higher than those in the non-sensitized rats respectively (p < 0.05, p < 0.01). In the laryngeal mucosa, the grade of eosinophilia in the sensitized rats was higher than that in the control (p < 0.01), but no significant differences of lymphocyte accumulation in the larynx between the two groups were found. Only a small number of eosinophil and lymphocyte accumulations in the trachea of the both groups were observed and no significant differences of the grade of inflammatory cells accumulation between the two groups were found. According to the results of this fundamental study, allergic laryngitis in Japanese cedar pollinosis might be originated from Japanese cedar pollen.

[A model for sensitivity determination of anticancer agents against chemical-induced colon cancer in rats].

Chemically-induced colon cancer was used to test the sensitivity of tumors to chemotherapeutic agents. Thirty-four Sprague-Dawley rats received dimethylhydrazine (40 mg/kg) s.c. once weekly for 10 weeks to induce colon cancer. Twenty weeks after beginning the carcinogen treatment, a barium enema was performed to determine the size of colon tumors. The animals were divided into CDDP group and CPT-11 group, in which the maximum tolerable dose of each drug was given. After 5 weeks of treatment, the barium enema was repeated. "Response" was assessed on the basis of tumor doubling time. Response rates in the CDDP and CPT-11 groups were 6% and 35%, respectively. This reflects the clinical data of those drugs and confirms the results of our previous study. The present study may be a predictive model for screening anticancer drugs for human colorectal malignancy.

Growth rate of experimental colon cancer in the absence of fecal stream and antitumor effect of UFT in rats.

Growth rate of a chemically-induced colon tumor in the absence of fecal stream was investigated and the tumors to a chemotherapeutic agent tested. Eighty Sprague-Dawley rats received dimethylhydrazine (40 mg/kg) s.c. once weekly for 10 wk to induce colon cancer. Then a colostomy was performed to produce a defunctioning colon without fecal stream. 22 wk after beginning the carcinogen treatment, a barium enema was performed to visualize tumors in the defunctionalized colon. 29 rats died postoperatively and 16 had no tumor radiographically. The remaining 35 rats were divided into a control group and UFT treatment group. After 5 wk of treatment, the barium enema was repeated. The mean doubling time of 19 tumors in the control group was 9.8 days +/- 4.0 (SD). Response to UFT was judged as effective when the doubling time exceeded 17.8 days, calculated from the mean +/- 2SDs in the control group. The response rate of UFT was 48%. The growth rate of colon tumors without fecal stream was faster and more stable than those with fecal stream; as a result, the sensitivity to UFT became higher than that in tumors with fecal stream (36%), which was reported in our previous study. The present experimental system may be more accurate for assessing the response to chemotherapeutic agents.

Effect of green tea rich in gamma-aminobutyric acid on blood pressure of Dahl salt-sensitive rats.

gamma-Aminobutyric acid (GABA) is known to be involved in the regulation of blood pressure by modulating the neurotransmitter release in the central and peripheral sympathetic nervous systems. This study investigated the antihypertensive effect of green tea rich in GABA (GABA-rich tea) in young and old Dahl salt-sensitive (S) rats. GABA-rich tea was made by fermenting fresh green tea leaves under nitrogen gas. In experiment 1, 21 11-month-old rats, fed a 4% NaCl diet for 3 weeks, were given water (group W), an ordinary tea solution (group T), or a GABA-rich tea solution (group G) for 4 weeks. The average GABA intake was 4.0 mg/rat per day. After 4 weeks of the treatment, blood pressure was significantly decreased in group G (176 +/- 4; P < .01) compared with group W (207 +/- 9) or group T (193 +/- 5 mm Hg). Plasma GABA levels were more elevated in group G (111 +/- 54) than in group W (not detectable) or group T (14 +/- 8 ng/mL; P < .01 v G). In experiment 2, 21 5-week-old rats, fed a 4% NaCl diet, were divided into groups W, T, and G. The average GABA intake was 1.8 mg/rat per day. Body weight or chow and beverage consumption did not differ significantly among the three groups. After 4 weeks of the treatment, although blood pressure was comparable in groups W and T (165 +/- 3 v 164 +/- 5 mm Hg, mean +/- SE), it was significantly lower in group G (142 +/- 3 mm Hg) than in the other groups (P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence analysis of the thyrotropin (TSH) receptor gene in congenital primary hypothyroidism associated with TSH unresponsiveness.

Congenital primary hypothyroidism due to thyrotropin (TSH) unresponsiveness is a very rare disorder and only a few cases have been documented previously. To elucidate whether structural abnormalities in the TSH receptor (TSHR) could be a primary underlying mechanism of this disorder, we analyzed nucleotide sequence of the entire coding region of the TSHR gene in three patients diagnosed with congenital primary hypothyroidism associated with TSH unresponsiveness. Diagnosis of TSH unresponsiveness was largely made based on the following criteria: (a) congenital primary hypothyroidism with autosomal recessive inheritance, (b) a nongoitrous thyroid gland in a normal position with low thyroidal radioactive iodine uptake, (c) normal in vitro TSH bioactivity or absent in vivo response to exogenous TSH, and (d) absence of thyroid autoantibodies. The TSHR cDNA was successfully obtained from RNA of peripheral mononuclear leukocytes with reverse transcription and polymerase chain reaction, and was sequenced directly. Comparison of these nucleotide sequences with the normal TSHR sequence revealed no difference in the predicted amino acid sequence with a heterozygous polymorphism in codon 601 in one patient, indicating absence of TSHR structural abnormalities in these patients. Our results indicate that congenital primary hypothyroidism associated with TSH unresponsiveness is unlikely to be due to mutations in the TSHR-structure gene.

Seasonal variations of nasal resistance in allergic rhinitis and environmental pollen counts. III: Efficacy of antiallergic agents.

We previously reported that, in a patient with Japanese cedar pollinosis, preseasonal increase in total nasal airway resistance (NAR) possibly caused from circannual endogenous rhythm was suppressed by preseasonal systemic administration of antiallergic agent. In this study, in order to determine the influence of preseasonal use of a topical antiallergic agent and seasonal administration of a peroral antiallergic agent, we made measurement of nasal airway resistance and pollen counts under the previous respective conditions in separated years. Preseasonal high mean values of total NAR for a couple of weeks was not suppressed by the preseasonal topical administration of ketotifen nasal spray in 1992 which was a low pollen season (974/cm2). However, the patient did not complained of severe episodes of hay fever at all throughout the season. Preseasonal increases in mean values of total NAR were observed in 1993 which was a high Japanese cedar pollen season (4,875/cm2) with NAR the same as untreated seasons. This may have been because the patient was not treated with preseasonal use of peroral azelastine. On extremely high Japanese cedar pollen count days further increased NAR occurred in the patient. The patient experienced relatively severe hay fever episodes, but used no other anti-histamine nor topical steroid during the season.

Overexpression of the intact thyrotropin receptor in a human thyroid carcinoma cell line.

Although thyrotropin is known to regulate thyroid cell differentiation and proliferation, human thyroid carcinoma cells are relatively insensitive or resistant to TSH stimulation. The expression levels of TSH receptor are significantly lower in carcinoma tissues than in normal tissues. Furthermore, in vitro human thyroid cell growth is not regulated by TSH itself. We, therefore, isolated neomycin-resistant stable human thyroid carcinoma cell (WRO cell) transfectants overexpressing intact human TSH receptor to evaluate the functional role of TSH receptor on carcinoma cells. Southern blot analysis confirmed incorporation and amplification of human TSH receptor complementary DNA sequences into genomic DNA. Northern gel analysis and reverse transcriptase-polymerase chain reaction analysis revealed the presence of specific TSH receptor messenger RNA (4.0 kilobases), and the specific binding and the affinity of [125I]TSH on stably transfected WRO cells were demonstrated compared to wild type. Nevertheless, impaired cAMP production to transfectants by TSH was observed. cAMP production was confirmed after stimulation of both wild type and transfectants by forskolin, cholera toxin, and isoproterenol. In contrast, TSH could affect the cytoplasmic calcium mobilization immediately after the addition of TSH to WRO transfectants. These results suggest that the impairment of TSH action on human thyroid carcinoma cells is not due to a major structural abnormality of the TSH receptor, reduction in the receptor number, or receptor affinity, but much more likely due to a TSH receptor-guanyl nucleotide-binding protein coupling defect.

Seasonal variations of nasal resistance in allergic rhinitis and environmental pollen counts. II: Efficacy of preseasonal therapy.

We gave Mao-bushi-saishin-to, a Chinese blended medicine, and azelastine to an adult patient with hay fever due to Japanese cedar pollen and measured nasal resistance and ambient floating pollen counts throughout the time of Japanese cedar pollination in separated years. In the patient Mao-bushi-saishin-to was effective against preseasonal increases in nasal airway resistance but could not control severe episodes of allergic rhinitis caused by high dose exposure to Japanese cedar pollen and also perhaps caused by a priming effect. Azelastine inhibited both pre- and post-seasonal increases in nasal airway resistance but not only on high pollen counts days.

[Intraperitoneal administration of recombinant interferon-beta for prevention in peritoneal metastasis of gastric cancer].

Recombinant interferon-beta (IFN-beta) shows a high anti-tumor effect on cancerous ascites, has little local irritative action and can safely be administered intraperitoneally. A randomized trial was conducted to evaluate the efficacy of intraperitoneal administration of IFN-beta for the purpose of preventing peritoneal metastasis of gastric cancer. The subjects selected were patients under 75 years of age who were macroscopically positive for serosal invasion and had undergone curative operation. Comparison was made by the envelope method between the cases given IFN-beta i.v. at 6 x 10(6) units continuously for 14 days from the day of operation and from 1 week after operation. MMC, 20 mg (i.v.) on the day of operation and 5-FU, 200 mg (p.o.) for 1 year from 2 weeks after operation were administered, respectively, in the two groups. The number of cases registered from March, 1987 to September, 1988 was 33, with 17 in the administration group and 16 in the control group. No difference was noted between the two groups in 3-year survival rate and disease free survival rate. As to the recurrence involving the peritoneum, however, there was a difference between the two groups, with 1 out of 6 cases in administration group and 3 out 4 cases in control group. Intraperitoneal chemotherapy can be an effective adjuvant therapy for gastric cancer, if cases are selected.

CGS 9896: agonist-antagonist benzodiazepine receptor activity revealed by anxiolytic, anticonvulsant and muscle relaxation assessment in rodents.

CGS 9896, a pyrazoloquinoline that potently binds to benzodiazepine receptors, has been reported to have anticonflict activity in conventional footshock paradigms and to antagonize pentylenetetrazol-induced seizures. In the present experiments, the pentylenetetrazol discriminative cue was blocked by CGS 9896 with a potency comparable to that of diazepam. CGS 9896 also selectively lengthened the latency to terminate self-initiated brain stimulation reward. These procedures extend the anxiolytic activity of CGS 9896 to models that do not rely upon footshock-induced conflict. CGS 9896 did not impair the traction reflex in mice, did not impair rotorod performance in rats, did not reduce unpunished operant responding and decreased motor activity only slightly, indicating no distinguishable sedation or muscle relaxation in rodent models. In fact, diazepam-induced rotorod impairment was blocked by CGS 9896. The anticonvulsant effects of CGS 9896, as indicated by audiogenic seizure and pentylenetetrazol-induced seizure studies, were substantial but were weaker than those of diazepam, possibly because of the muscle relaxant component of diazepam. Ethanol-induced motor impairment was potentiated more markedly by diazepam than by CGS 9896. Mixed agonist-antagonist properties of CGS 9896 therefore emerge when a comprehensive battery of behavioral assessments is utilized. CGS 9896 may have clinical anxiolytic activity without sedation or muscle relaxation.