Effects of fermented soymilk with Lacticaseibacillus paracasei YIT 9029 on gut microbiota and defecation habits: a randomised, double-blind, placebo-controlled study.

Previous studies have demonstrated that soymilk and Lacticaseibacillus paracasei YIT 9029 (strain Shirota: LcS) each beneficially affect the gut microbiota and defecation habits. To investigate the effects of daily consumption of fermented soymilk containing LcS (FSM), we conducted a randomised, double-blind, placebo-controlled study of 112 healthy Japanese adults with a low faecal Bifidobacterium count. They consumed 100 ml FSM or placebo (unfermented soymilk base) once daily for 4 weeks. Their gut microbiota was analysed by 16S rRNA gene amplicon sequencing and quantitative reverse transcription-polymerase chain reaction (PCR), and faecal short-chain fatty acids (SCFAs) and urinary putrefactive products were assessed during the pre- and post-consumption periods. Defecation habits were examined weekly using a subjective questionnaire. In the post-consumption period, living LcS were not detected in two subjects in the FSM group (n = 57) but were detected in one subject in the SM group (n = 55). The FSM group had a significantly higher number and relative abundance of faecal lactobacilli compared with the placebo group. The relative abundance of Bifidobacterium, alpha-diversity of microbiota, and concentrations of acetate and total SCFAs in faeces were significantly increased in the FSM group, although no significant differences were detected between the groups. The number of defecations and defecation days per week significantly increased in both groups. Subgroup analysis of 109 subjects, excluding 3 with inconsistent LcS detection (2 and 1 subjects in the FSM and SM groups, respectively), revealed that the FSM group (n = 55) had significantly greater increases in faecal acetate concentration compared with the SM group (n = 54) and significant upregulation of pathways related to energy production or glucose metabolism in the gut microbiota. These findings suggest that daily FSM consumption improves the gut microbiota and intestinal environment in healthy adults and may help to maintain health and prevent diseases. Registered at the University Hospital Medical Information Network (UMIN) clinical trials registry under: UMIN 000035612.

Gamma-Ray Observation of the Cygnus Region in the 100-TeV Energy Region.

We report observations of gamma-ray emissions with energies in the 100-TeV energy region from the Cygnus region in our Galaxy. Two sources are significantly detected in the directions of the Cygnus OB1 and OB2 associations. Based on their positional coincidences, we associate one with a pulsar PSR J2032+4127 and the other mainly with a pulsar wind nebula PWN G75.2+0.1, with the pulsar moving away from its original birthplace situated around the centroid of the observed gamma-ray emission. This work would stimulate further studies of particle acceleration mechanisms at these gamma-ray sources.

Bifidobacterium bifidum YIT 10347 fermented milk exerts beneficial effects on gastrointestinal discomfort and symptoms in healthy adults: A double-blind, randomized, placebo-controlled study.

In a preliminary open-label trial by our group, Bifidobacterium bifidum YIT 10347 (YIT10347) relieved gastric symptoms in patients with functional gastrointestinal disorders. Hence, in this study, we investigated the effects of YIT10347 on gastrointestinal symptoms in healthy adults. In this prospective double-blind, randomized, placebo-controlled trial (UMIN000024654), 100 healthy Japanese adults were randomly assigned to a YIT10347 group or placebo group and consumed 100 mL of YIT10347-fermented milk or placebo fermented milk, respectively, every day for 4 wk. Gastrointestinal symptoms were evaluated by using the modified Frequency Scale for Symptoms of Gastroesophageal Reflux Disease (m-FSSG) and Gastrointestinal Symptom Rating Scale (GSRS) as primary endpoints. Mental symptoms, quality of life, salivary stress markers, and gastric emptying were evaluated as secondary endpoints. Effectiveness and safety were analyzed in a per-protocol set (YIT10347 group, n = 39; placebo group, n = 40) and full analysis set (YIT10347 group, n = 50; placebo group, n = 50), respectively. In the m-FSSG evaluation, the YIT10347 group had a significantly higher relief rate of postprandial discomfort and greater changes in postprandial epigastric pain score from baseline than the placebo group. In the GSRS evaluation, the YIT10347 group had significantly higher relief rates of overall gastrointestinal symptoms, upper gastrointestinal symptoms, flatus, and diarrhea than the placebo group. We detected no significant differences in scores or relief rates of mental symptoms and quality of life, a salivary stress marker, or gastric emptying between the 2 groups. No severe adverse events associated with test beverage consumption were observed in either group. These findings suggest that daily consumption of YIT10347-fermented milk exerts beneficial effects on gastrointestinal discomfort and symptoms such as postprandial discomfort and epigastric pain in healthy adults.

Living cells of probiotic Bifidobacterium bifidum YIT 10347 detected on gastric mucosa in humans.

The probiotic strain Bifidobacterium bifidum YIT 10347 has been demonstrated to inhibit Helicobacter pylori activity, prevent injury to the gastric mucosa, and improve general gastric malaise symptoms in H. pylori positive patients. This study aimed to investigate the adhering activity and localisation of B. bifidum YIT 10347 to gastric cells and tissue in vitro, and in human in vivo to clarify the mechanism of its beneficial effects on the stomach. The in vitro study found the adhesion rate of B. bifidum YIT 10347 to human gastric epithelial cells was about 10 times higher than that of lactic acid bacteria and other bifidobacteria. In the human study, 5 H. pylori negative and 12 H. pylori positive subjects ingested milk fermented with B. bifidum YIT 10347. B. bifidum YIT 10347 cells were measured by RT-qPCR for in gastric biopsy samples. Living B. bifidum YIT 10347 cells were detected in the biopsy samples in H. pylori negative subjects (105 cells/g and 104 cells/g at 1 h and 2 h after ingestion, respectively) and H. pylori positive subjects (104 cells/g at 1 h after the ingestion). Moreover, immunostaining analysis of tissue sections found that B. bifidum YIT 10347 cells were located at the interstitial mucin layer of the stomach. These results suggest that cells of probiotic B. bifidum YIT 10347 adhered to the human gastric mucosa in a live state, and that the higher adhering activity of B. bifidum YIT 10347 to the gastric mucosa may be involved in its beneficial effects on the human stomach.

Health benefits of fermented milk containing Bifidobacterium bifidum YIT 10347 on gastric symptoms in adults.

We conducted a preliminary open trial (trial 1) and a double-blind, placebo-controlled, crossover trial (trial 2) to examine how fermented milk containing the probiotic Bifidobacterium bifidum YIT 10347 affects gastric and lower abdominal symptoms in adults taking no medication. In trial 1, subjects with or without gastric and lower abdominal symptoms ingested fermented milk containing B. bifidum YIT 10347 daily for 2 wk. In trial 2, subjects with gastric symptoms ingested fermented milk containing B. bifidum YIT 10347 (active preparation) or placebo daily for 2 wk, followed by crossover for 3 wk after a washout period. Before (baseline) and 1 and 2 wk after ingestion, subjects completed a questionnaire. In trial 1 (305 subjects), the prevalence of gastric and lower abdominal symptoms was 46 and 58%, respectively, at baseline. Ingestion of B. bifidum YIT 10347 significantly decreased the prevalence of gastric and lower abdominal symptoms from 45 to 33% at 1 wk and to 28% at 2 wk, and from 57 to 40% at 2 wk, respectively. In subjects with gastric symptoms at baseline, the average gastric symptom score per subject significantly decreased by 0.9 at 1 wk and 1.2 at 2 wk. In trial 2 (27 subjects), ingestion of the active preparation significantly decreased the average gastric symptoms score per subject by 1.0 at 1 wk and 1.1 at 2 wk, but ingestion of placebo milk had no effect. No side effects were reported by any subjects in either trial. We conclude that fermented milk containing B. bifidum YIT 10347 has the potential to provide health benefits by alleviating gastric symptoms in subjects taking no medication.

Effect of Bifidobacterium bifidum BF-1 on gastric protection and mucin production in an acute gastric injury rat model.

Homeostasis in the stomach environment is maintained by the balance of protective factors such as gastric mucus and aggressive factors such as gastric acid, stress, alcohol, and drugs. An overload of aggressive factors that upsets this balance can induce gastric injury. Fermented milk that contains Bifidobacterium bifidum BF-1 (BF-1), a probiotic strain, and Streptococcus thermophilus YIT 2021 (ST) is known to improve Helicobacter pylori-associated gastritis in humans. Here, we investigated the gastroprotective potential of BF-1 in a rat model of acid-ethanol-induced acute gastric injury to fully elucidate its potential compared with ST. Living BF-1, ST, or vehicle was orally administrated to rats, and acid-ethanol gastric injury was induced 2h later. The gastric injury rate was determined and shown to be significantly lower in the BF-1 group than in the vehicle group, which showed a similar level to the ST group. The production of gastric mucin and the expression of several target genes associated with protection and inflammation were examined before and after induction of gastric injury. Interestingly, mucin 5ac (muc5ac) gene expression in gastric corpus samples and gastric mucin production in stomach samples from the BF-1 group, but not the ST group, were significantly higher than those in the respective samples from the vehicle group. These findings indicate that BF-1 has the potential to provide gastroprotection, alleviating acute gastric injury by enhancing the production of gastric mucin in a rat model.

[Relation between intraoperative salvaged blood transfusion and postoperative infection after cardiac surgery].

In cardiac surgery, several studies have shown bacterial contamination rates of intraoperative salvaged blood ranging from 12.7 to 96.8%. We evaluated the relation between intraoperative salvaged blood transfusion produced by the Cell Saver 5 device (Haemonetics Corp., Braintree, MA, USA) and postoperative infection determined by bacteriological study and the postoperative clinical course after cardiac surgery. Seven cases of cardiac surgery were investigated by bacteriological study. Although bacteria were cultured from all salvaged blood, no bacteria were cultured from the patients' blood 24 hours after salvaged blood infusion. Another 26 patients who underwent cardiac surgery, were divided into groups: group CS (n = 15) with salvaged blood transfusion after operation and group N (n = 11) without salvaged blood transfusion, and were evaluated in relation to the postoperative clinical course. There were no statistically significant differences between group CS and group N in the data of WBC, CRP and maximum body temperature. One case of deep sternal wound infection and 2 cases of local wound infection were observed in group CS, but none in group N (p = 0.18). These complications were treated by primary closure without muscle flaps. We conclude that salvaged blood autotransfusion was not related to postoperative infections in cardiac surgery.

[Bacterial contamination of salvaged blood in open heart surgery: is that an airborne contamination or a normal skin flora contamination?].

We investigated sources of bacterial contamination of intraoperative salvaged blood producted by autologous transfusions device (CS; CELL SAVER 5, Heamonetics Corp., Braintree, MA). Eleven patients undergone open heart surgeries including 2 emergency operations with a median sternotomy enrolled in this study. Blood samples were drawn from salvaged blood bags. Airborne contaminants (AB) were collected by a blood agar plate put besides the operation bed for 30 minutes. The median wounds samples were collected by a swab. Bacterial growth was detected in 81.8% of salvaged blood samples. Twenty-nine bacterium were isolated from CS, 72.4% of those were Staphylococci. 9.1% of sample was positive in wound swabs. Forty bacterium were isolated from plate cultures. 65% of them were Staphylococci. Staphylococcus epidermidis and coagulase negative Staphylococcus isolated both CS and AB in the 2 cases had the same identify codes, and incubated from several AB cultures. Corynebacterium sp. is also isolated from both CS and AB cultures in other 2 same cases. In 7 out of 8 cases (87.5%), from which Staphylococci isolated in CS, the Staphylococci were cultured from AB in not the same but the other cases. In conclusion, highly incidence of the identification in identical code of Staphylococci indicated that the main source of CS contamination was highly suspected to AB.

Oblique aortic valve replacement and coronary artery bypass grafting for severely calcified narrow aortic root with unstable angina.

We report an 84-year-old woman diagnosed with aortic stenosis and regurgitation with a severely calcified narrow aortic root and left main coronary artery trunk stenosis with triple-vessel coronary artery disease. Emergency aortic valve replacement and triple coronary artery bypass grafting were successful. The aortic annulus was small and heavily calcified, and the ascending aorta, the sinus of valsalva and the anterior leaflet of the mitral valve were severely calcified. A St. Jude Medical valve 19A (St. Jude Medical Inc., St. Paul, MN) was inserted obliquely along the noncoronary sinus. This technique is a useful alternative in cases where the patient's life is at risk in situations involving severe extensive calcification of a narrow aortic root.

[Maze procedure for atrial fibrillation associated with aortic valve disease in an elderly patient: report of a case].

The choice of a valve substitute in elderly patients remains controversial. Moreover, the combined use of the maze procedure in elderly patients raises further discussion. We report a case of bioprosthetic aortic valve replacement concomitant with the maze procedure in a 74-year-old man. With symptom of orthopnea and short of breath, he was diagnosed as aortic stenosis and regurgitation with atrial fibrillation. We performed the bioprosthesis aortic valve replacement and the maze procedure in this patient. He was extubated next day and discharged on the fifteenth day after the operation without any complications. Postoperatively he gained a sinus regular rhythm and A wave of transmitral flow was detected by Doppler echocardiography. Now, he enjoys his better quality of life without any drugs including warfarin. Although combined aortic valve replacement and maze procedure are more invasive for elderly patients, these procedures provides better quality of life after the operation.

[Is open heart surgery clean: bacteriologic analysis of salvaged blood].

In this prospective study, we evaluated contamination of surgical fields in open heart operations by salvaged blood culture used in autologous transfusions device (Cell Saver 5, Heamonetics Corp., Braintree, MA, USA). And also, we prospectively investigated an efficacy of pre- and intra-operative prophylactic antibiotics administrations (cefazolin). Thirty patients undergone open heart surgeries with median sternotomy enrolled in this study. The patients were divided into two groups, group A (n = 15); without prophylactic antibiotics administration, group B (n = 15); with pre- and intra-operative prophylactic antibiotics administrations. Blood samples were drawn through the right atrium after the discontinuation of CPB and from salvaged blood bags. Bacterial growth was detected in 80.0% of salvaged blood samples in group A, 86.7% in group B (p = 0.62). Whereas no bacterial growth detection in blood samples though the right atrium. Quantitative estimates of contaminations showed 1.06 +/- 1.41, 0.90 +/- 1.24 cfu/ml, respectively (p = 0.22). Although bacterial growth rate were not statistically significant difference between groups, detective rate of Staphylococci was remarkably decreased (p = 0.005) in group B. Pre- and intra-operative prophylactic antibiotics administrations were effective for Staphylococci, but not whole microorganisms. In conclusion, salvaged blood used in autologous transfusions was highly contaminated and it suggests that surgical fields were not clear. Prophylactic antibiotics administrations were effective especially for Staphylococci.

[A successful case of hybrid therapy for the left main trunk and triple coronary vessel lesions with acute myocardial infarction and cardiogenic shock].

A 86-year-patient who had acute myocardial infarction and critical cardiogenic shock was diagnosed to have the left main trunk (LMT) and triple vessel disease. Emergent coronary artery bypass grafting to the left anterior descending artery was performed using saphenous vein graft without cardiopulmonary bypass through median sternotomy. On the 41st postoperative day, catheter intervention was performed to the remaining lesions by stenting of LMT and percutaneous transluminal coronary angioplasty to the right coronary artery lesions. Tl scintigraphy showed remarkable reduction of myocardial ischemia. Hybrid therapy is the effective new strategy for critical cases which cannot be successfully and securely treated by medical or surgical approach alone.

Changing profile of abdominal wall defects in Japan: results of a national survey.

BACKGROUND/PURPOSE: The incidence of gastroschisis has increased over the past 3 decades in a number of countries. To elucidate the Japanese status of anterior abdominal wall defects, the Japanese Society of Pediatric Surgeons conducted a national survey in Japan. METHODS: Information was obtained by sending out a questionnaire to 192 University Hospitals, Children's hospitals, and general hospitals that each had more than 200 beds. The characteristics of the patients including the birth date, birth weight, gestations, rate of associated anomalies, rate of antenatal diagnosis and prognosis, maternal age, gravidity, history of smoking, and drug use were analyzed. RESULTS: The authors obtained answers from 149 institutions, including 1,785 cases of omphalocele and 970 cases of gastroschisis, which were treated between 1975 to 1997. There was a significant increase in the incidence of gastroschisis, from 0.131 in 1975 to 1980, 0.269 in 1981 to 1985, 0.337 in 1986 to 1990, 0.461 in 1991 to 1995 to 0.467 per 10,000 births in 1996 to 1997. The incidence of omphalocele was 0.322 in 1975 to 1980, 0.567 in 1981 to 1985, 0.657 in 1986 to 1990, 0.741 in 1991 to 1995 to 0.626 per 10,000 births in 1996 to 1997, respectively. In the omphalocele group, 43.1% of the mothers were between 25 to 29 years of age, whereas in the gastroschisis group 42.6% of the mothers were 20 to 24 years of age. In the gastroschisis group, the number of primipara mothers was larger than that of multipara mothers. In the omphalocele group, approximately 10% of the mothers smoked during each period, whereas in the gastroschisis group, the percentage of smoking mothers increased chronologically from 12.9% in 1981 to 1985, 18.7% in 1986 to 1990, 23.5% in 1991 to 1995 and 29.3% in 1996 to 1997. A history of drug use by the mother was approximately 10% for both the omphalocele and gastroschisis groups. In the omphalocele group, 55.9% had associated anomalies against 21.8% in the gastroschisis group. Approximately 10% in the omphalocele group and less than 3% in the gastroschisis group showed chromosomal abnormalities. From 1986, a significant number of cases detected antenatally has been observed. CONCLUSIONS: There have been substantial changes in the incidence of anterior abdominal wall defects, particularly regarding gastroschisis in Japan. The reasons for such changes are most likely multifactorial, further epidemiological monitoring is thus called for.

Physiological changes in Pachinko players; beta-endorphin, catecholamines, immune system substances and heart rate.

Pachinko is a popular form of recreation in Japan. However, in recent years, along with Pachinko's popularity, "Pachinko dependence" has become topical news. The purpose of this study was to investigate beta-endorphin, catecholamines, immune system responses and heart rate during the playing of Pachinko. The following significant results were observed. (1) Plasma concentration of beta-endorphin increased before playing Pachinko and while in the Pachinko-center (p < 0.05). (2) Beta-endorphin and norepinephrine increased when the player began to win (i.e. at "Fever-start") compared to baseline (p < 0.05). (3) Beta-endorphin, norepinephrine and dopamine increased when the winning streak finished (i.e. at "Fever-end") compared to baseline (p < 0.05-0.01). (4) Norepinephrine increased past 30 minutes after "Fever-end" compared to baseline (p < 0.05). (5) Heart rate increased before "Fever-start" compared to baseline, peaked at "Fever-start" and rapidly decreased to match rates measured at rest. But the increase was observed from 200 seconds after "Fever-start" (p < 0.05-0.001). (6) There was a positive correlation between the number of hours subjects played Pachinko in a week and the differences between beta-endorphin levels at "Fever-start" and those at rest (p < 0.05). (7) The number of T-cells decreased while the number of NK cells increased at "Fever-start" compared to baseline (p < .05). These results suggest that intracerebral substances such as beta-endorphin and dopamine are involved in the habit-forming behavior associated with Pachinko.

[Efficacy and safety of a single oral dose of pilsicainide in supraventricular arrhythmia after coronary artery bypass grafting].

We evaluated the efficacy and safety of administration of a single oral dose of Pilsicainide, a class Ic antiarrhythmic agent, in 34 patients (26 men, 8 women, age from 48 to 81, mean age 66 +/- 8 years) who developed supraventricular arrhythmia after coronary artery bypass grafting (CABG). A total of 42 episodes of postoperative supraventricular arrhythmia, with the majority occurring 2-4 days after CABG, were classified as follows: paroxysmal atrial fibrillation, 34; paroxysmal atrial flutter, 6; and sinus tachycardia, 2. Sinus rhythm was restored in 32 episodes (78%) by treatment with oral pilsicainide given in a single dose of 50 ro 100 mg. Successful conversion was obtained within 90 minutes in 44% and 3 hours in 56% of episodes, with a mean conversion time of 119.2 +/- 107.5 minutes after the administration of pilsicainide. The mean conversion times tended t highlight the difference between the 50 mg and 100 mg doses, but this difference was not statistically significant (p = 0.053). The ventricular rate was significantly reduced from 123.3 +/- 29.5 beats/min to 85.6 +/- 19.9 beats/min (p < 0.0001), however no significant changes in blood pressure or no significant side effects were observed. The results of the present study suggest that a single oral dose of pilsicainide, administered for its favorable pharmacokinetic profile and lack of hemodynamic side effects, is a valuable drug for converting supraventricular arrhythmia to sinus rhythm in most patients after CABG.

Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues.

Expression of the multidrug-resistance protein gene MRP, which confers non-P-glycoprotein-mediated multidrug resistance, has been found in many drug-resistant variants and tumor samples. Recent studies have demonstrated that MRP functions as an ATP-dependent transporter functionally related to the previously described glutathione-conjugate (GS-X) pump. We have shown recently that the MRP and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit mRNA levels are coordinately overexpressed in cisplatin (CP)-resistant human leukemia cells (Ishikawa et al., J Biol Chem 271: 14981-14988, 1996) and frequently co-elevated in human colorectal tumors (Kuo et al., Cancer Res 56: 3642-3644, 1996). In the present study, we showed the coexpression patterns of thirteen additional human drug-resistant cell lines representing different tumor cell origins selected with different agents, except for one doxorubicin-selected line which demonstrated minor elevation in MRP mRNA with no detectable increase in gamma-GCS mRNA, suggesting that the increase of MRP mRNA preceded the increase in gamma-GCS mRNA. Furthermore, in seventeen randomly selected untreated tumor cell lines, the overall correlation coefficient between MRP and gamma-GCS mRNA levels was 0.861. In normal mice, the correlation coefficient of mrp and gamma-gcs mRNA was 0.662 in fourteen tissues (kidney and liver were not included) analyzed. Kidney and liver expressed low levels of mrp relative to gamma-gcs; however, these two tissues expressed high levels of a functionally related mrp homologue, mrp2 (cMoat or cMrp), which may have compensated for the underexpressed mrp in maintaining the total GS-X pump activities. Altogether, these results demonstrated the frequent coexpression of these two genes in various cell settings.

[Hemodynamic evaluations of patients with small aortic annulus with St. Jude Medical prosthetic heart valve].

As the elderly population in Japan increases, senile degenerative aortic valvular disease also tends to increase. These patients often have a small aortic annulus. The problem of "valve-patient-mismatch" occurs when a small prosthesis is inserted into a patient with a small aortic annulus. To avoid annular enlargement after aortic valve replacement (AVR), we tried to use a small-sized St. Jude Medical (SJM) valve. From September 1988 through November 1996, 110 AVR were performed in our institution. In these cases, 30 underwent AVR with a small sized SJM valve (male < or = 21 mm, female < or = 19 mm). Dobutamine stress echocardiography was performed in 19 patients who had undergone AVR with a small-sized SJM valve. Surgical results were also compared between patients with small aortic annulus and those with normal-sized aortic annulus. Using Doppler echocardiography, pressure gradients (PG), cardiac index (CI), effective orifice area (EOA), and performance index (PI) were calculated at rest and during stress. The mean body surface area (BSA) of patient who had undergone AVR with SJM19A, 19HP and 21A was 1.40, 1.42 and 1.56 m2, respectively. With dobutamine stress, heart rates, PG and CI increased significantly. Mean and maximum PG of patients with 19HP (8.0 and 15.4 mmHg at rest, 12.9 and 28.0 mmHg under stress, respectively) and 21A (9.5 and 19.1 mmHg at rest, 16.5 and 35.3 mmHg under stress, respectively) were relatively low. EOA index (EOAI) of patient with 19HP showed the highest values mean 0.93 cm2/m2. PIs tended to be higher with HP models than with standard models. The tests were completed without significant side effects such as frequent ventricular arrhythmias. Among the cases with small aortic annulus, there were no operative deaths or hospital deaths. There were also no late deaths, episodes of hemorrhage or thrombosis. Conclusions. In our institution, AVR was performed safely without any aortic annular enlargement with a small aortic anulus in small BSA patients. Postoperative hemodynamic data obtained by echocardiography were satisfactory for all patients at rest and even during maximum dobutamine stress test.

Antitumor effect of hepatocyte growth factor on hepatoblastoma.

A six month-old girl presented with an abdominal mass, and high serum level of alpha-fetoprotein. She was diagnosed as having a well-differentiated hepatoblastoma by open biopsy. The biopsy specimen was transplanted on a nude mouse, and a xenograft was successfully established. Because the xenograft maintained the characteristics of the original tumor, the effect of hepatocyte growth factor (HGF) on hepatoblastoma xenograft was investigated. Recently HGF was reported to be involved in growth, invasion, and metastasis of tumor cells. Contrary to our expectations, the treatment of hepatoblastoma xenograft with recombinant 20 ng/ml HGF produced a marked inhibition of cell growth and a suppression of producing alpha-fetoprotein.

Transient induction of the MRP/GS-X pump and gamma-glutamylcysteine synthetase by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea in human glioma cells.

Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), an alkylating antitumor agent the primary target of which has been thought to be DNA, resulted in elevated expression of mRNA for multidrug resistance-associated protein (MRP) within the first 2 h and then a decrease in expression 24 h after the treatment. Western blot analyses revealed that levels of MRP in these ACNU-treated cells paralleled mRNA levels. Membrane vesicles prepared from ACNU-treated cells also displayed elevated transport activities for leukotriene C4, a known substrate for MRP. Gamma-glutamylcysteine synthetase (gamma-GCS) mRNA expression was coinduced with MRP by ACNU. Because gamma-GCS is the rate-limiting enzyme involved in the de novo biosynthesis of glutathione, increases in glutathione were also transiently induced by ACNU. These results demonstrate for the first time that the expression of functional MRP and gamma-GCS can be transiently coinduced by ACNU. Multiple short exposures (1 h) of ACNU following a long duration (1 week) of drug-free conditions resulted in the development of an ACNU-resistant population (designated A172R) that overexpressed MRP/gamma-GCS mRNA and had elevated transport activities for leukotriene C4. A172R exhibited cross-resistance to the antitumor drug doxorubicin and heavy metal sodium arsenate but not to cisplatin. Our results also demonstrate that intermittent treatments of human glioma cells with ACNU can lead to the development of MRP-related multidrug resistance. These results, taken together, reveal a possible new mechanism of the development of drug resistance for the antitumor nitrosoureas.

Posttranscriptional regulation of MRP/GS-X pump and gamma-glutamylcysteine synthetase expression by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea and by cycloheximide in human glioma cells.

Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethy-3-nitrosourea (ACNU) for 2 to 4 hr resulted in a 2- to 3-fold increase in steady-state levels of multidrug resistance-associated protein (MRP) and gamma-glutamylcysteine synthetase (gamma-GCS) mRNA. Nuclear run-on assays revealed a less than 0.5-fold increase in transcription rates of these genes under the same treatment conditions, suggesting that posttranscriptional regulation plays an important role for the increased mRNA levels. In the absence of ACNU, rates of MRP and gamma-GCS mRNA degradation were similar in A172 cells as determined by incubating cells with the RNase inhibitor, Actinomycin D. ACNU treatments resulted in increased MRP mRNA stability. Induction of MRP and gamma-GCS mRNA by ACNU apparently did not require de novo protein synthesis as determined by the use of protein synthesis inhibitor cycloheximide (CHX). However, CHX alone could induce accumulation of gamma-GCS mRNA, also by posttranscriptional mechanism. Taken together, these results demonstrate that (i) posttranscriptional regulation is primarily involved in the induction of MRP and gamma-GCS expression by ACNU and CHX in human glioma cells; and (ii) despite the fact that these two genes have been reported to be frequently co-expressed, their responses to the treatments of RNA and protein synthesis inhibitors are not the same.