Establishment and molecular characterization of a novel leukemic cell line with Philadelphia chromosome expressing p230 BCR/ABL fusion protein.

The cell line AR230 was established from the peripheral blood mononuclear cells of a patient with chronic myeloid leukemia and t(9;22) translocation bearing a variant type of BCR/ABL rearrangement. AR230 expresses a BCR/ABL fusion protein with a molecular mass of 230 kilodaltons (kDa) due to the insertion of 180 amino acids encoded by 3' exons of BCR (b4 to c3). An immune complex kinase assay showed that the 230-kDa BCR/ABL protein ahd autophosphorylation activity. Immunoprecipitation analysis revealed a stable complex of GRB2 and 230-kDa BCR/ABL proteins, indicating that the Ras activation pathway is involved in the process of transformation. AR230 expressed another short transcript consisting of a BCRc2/ABL junction, which is associated with a stop signal shortly after the junction. To our knowledge, this is the first cell line expressing a 230-kDa fusion product of BCR/ABL. AR230 will be useful for studying the biological function of divergent BCR/ABL proteins.

Salt consumption and nutritional state especially dietary protein level.

To examine the background of excessive salt intake by the people who live in the northeastern district of the mainland of Japan, statistical analysis was carried out on salt consumption with nutritional status with data from the National Nutrition Survey of Japan and prefectural reports. We found that the dietary protein level (using the index animal-protein calorie intake:total calorie intake) was inversely proportional to the salt intake. To clarify the relationship between taste preference and nutritional status, the effects of dietary protein levels on the preference for salt were studied in spontaneously hypertensive rats (SHR), Wistar-Slc rats, and Sprague-Dawley (SD) rats. We found that the appetite or preference for sodium chloride depends not only on a genetic factor but also on the nutritional status, ie, dietary protein levels. We also observed that the preference for the monosodium glutamate solution was not induced in animals fed a low-protein diet. From these observations we speculated that the threshold of taste sensation is increased in rats fed a low-protein diet. To determine the threshold of taste sensation for NaCl, we calculated the turnover time of taste buds, which was lengthened in rats fed a low-protein diet. The findings from statistical analysis and the results from animal experiments suggest that taste preference is influenced not only by genetic factors but also by the nutritional status. Our hypothesis is that dietary protein level is one of the important factors affecting the taste preference for sodium chloride.

Acetazolamide specifically inhibits lingual trigeminal nerve responses to carbon dioxide.

The goal of this study was to examine the role of the enzyme, carbonic anhydrase, in oral trigeminal chemoreception with particular regard to the reception of CO2. Using both single and multiunit recordings of trigeminal neurons in the lingual nerve of rat, we measured responses to cool (24 degrees C), noxiously hot (55 degrees C) and cold (8 degrees C) H2O, NH4Cl and supersaturated solutions of CO2 (24 degrees C and 33 degrees C). The importance of peripheral carbonic anhydrase was tested by inhibiting enzyme activity with acetazolamide (15 mg/kg b.w.). Single unit responses to CO2 and HCl suggest that neural sensitivity to CO2 is not simply a function of extraepithelial pH. Responses to CO2 were significantly inhibited by acetazolamide while the responses to thermal stimuli and NH4Cl were not. The results support a role for carbonic anhydrase in trigeminal responses to CO2. Furthermore, the results suggest that intraepithelial acidification mediated by carbonic anhydrase may be the basis for sensitivity to CO2.

Nutritional studies of the roles of dietary protein levels and umami in the preference response to sodium chloride for experimental animals.

Previously we found that appetite or preference for sodium chloride depends not only on a genetic factor, but also on the nutritional status, i.e., dietary protein levels. To determine whether all kinds of protein had reducing effects on sodium chloride intake, the effect of dietary protein source on appetite or preference for sodium chloride was studied. It was found that the effect depended not only on protein levels, but also on the protein sources, i.e., amino acid patterns of diets. On the other hand, the turnover time of gustatory taste bud cells was measured by the pulse-labeled method using 3H-thymidine. It was found that the turnover time of gustatory cells in the taste buds of rats fed a low-protein diet was longer than in rats fed a high-protein diet. This suggested that some reduction of receptor functions may occur with low-protein diet. It was found that the dietary protein levels and umami also modified appetite or preference for alcohol solutions.

Zinc deficiency and taste dysfunction; contribution of carbonic anhydrase, a zinc-metalloenzyme, to normal taste sensation.

The present study was designed to clarify the effect of zinc deficiency on sodium chloride preference, the lingual trigeminal and taste nerves transduction, and carbonic anhydrase (CA) activity of the tongue surface and salivary gland. Male SD rats, 4 weeks old, were divided into four groups, and fed zinc-deficient (Zn-Def), low-zinc (Low-Zn), and zinc-sufficient diets with free access (Zn-Suf) and pair-feeding (Pair-fed). After taking part in the preference tests for 42 days, the rats were provided for the chorda tympani and lingual trigeminal nerves recordings, then finally sacrificed and the tongue and submandibular gland excised to measure CA activity. Sodium chloride preference increased only after 4 days of the feeding of zinc-deficient and low-zinc diets, which means that the taste abnormality appears abruptly in zinc deficieny and even though in marginal zinc deficiency. Reduced CA activities of the taste-related tissues in zinc-deficient group paralleled well with the decreased taste and lingual trigeminal nerves sensitivities.

Effects of restricted diet and intestinal flora on the life span of small intestine epithelial cells in mice.

Previous data have shown that the life span of small intestine epithelial cells in germ-free (GF) mice was 4.3 days, while that in conventional (CV) mice was 2.1 days, under ad libitum feeding. On the other hand, in the author's laboratory, it was also found that feeding conditions affected the cells' life span. That is, in CV mice the life span of the cells lengthened under restricted feeding (2.6 days), compared with under and libitum feeding (1.8 days). In the present experiment the life span of small intestine epithelial cells was investigated using radioautography, under controlled feeding (setting it equal to ad libitum feeding) and restricted feeding, in both CV and GF mice. Small intestine samples were taken from the middle part of duodenum, jejunum and ileum. Body weight changes, organ wet weights and intestine were also measured. In the lower part of the small intestine the effects of a restricted diet on epithelial cell life span prolongation appeared clearly in CV mice, but this effect was reduced in GF mice. This may be partly because the restricted group had slightly shorter villi in the case of GF mice.

Gastrointestinal responses to graded levels of cellulose feeding in conventional and germ-free mice.

Conventional (CV) and germ-free (GF) mice were fed on a semi-synthetic diet containing graded levels of cellulose (0, 5, 15 and 30%), and thereafter the length and wet weight of intestine, the morphology of mid-jejunum epithelium and the turnover of mid-jejunum epithelial cells were determined. The following results were obtained. enlarged stomachs were observed in CV mice fed on a non-cellulose or 30% cellulose diet, but there were no differences found among the four dietary groups in GF mice. On the other hand, no effect of intestinal bacteria was observed, at least with regard to caecum weight, since the responses of caecum wet weight to the graded cellulose intake in CV mice were similar to those in GF mice. The responses of intestine length to graded cellulose intake differed between CV and GF mice, indicating that intestinal bacteria may modify the length of intestine in some way. Marked differences were observed in the responses of villus length to graded levels of cellulose between CV and GF mice. That is, in CV mice there was a gradual increase in villus length as cellulose content increased, whereas in GF mice there was a marked decrease in villus length. In the CV mice, graded levels of dietary cellulose had no effect on the epithelial cell turnover. On the other hand, in the GF mice it was observed that the greater the amount of dietary cellulose, the faster the turnover rate becomes. As a result, dietary cellulose would enhance the turnover rates of jejunal epithelial cells only in the absence of intestinal bacteria.

Menaquinone-4 accumulation in various tissues after an oral administration of phylloquinone in Wistar rats.

The distributions of phylloquinone (PK) and menaquinone-4 (MK-4) in various tissues were assessed after the oral administration of phylloquinone. Wistar rats were fed a vitamin-K-deficient diet for nine days, fasted for 24 h and then given phylloquinone orally at 4 mg/kg body weight. Rats were sacrificed 0, 6, 12 and 24 h after the administration, and an analysis was made of the vitamin K analogues in the plasma, liver, brain, testis, kidney and spleen. The phylloquinone concentration in plasma and the tissues reached a peak 6 h after the oral administration of phylloquinone. By contrast, the concentration of MK-4 peaked in the liver, plasma, kidney and spleen at 12 h, and in brain and testis at 24 h. This data suggests that the ingested phylloquinone was probably converted into MK-4 within the tissues themselves, rather than via hepatic metabolism. The evidence for this is that, after phylloquinone administration, (i) in each of the tissues, the MK-4 concentration increased much more slowly than that of phylloquinone, and (ii) the MK-4 concentration in the plasma and liver reached only much lower levels than those seen in other tissues.

A high biotin diet improves the impaired glucose tolerance of long-term spontaneously hyperglycemic rats with non-insulin-dependent diabetes mellitus.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, serving as a spontaneously diabetic model with non-insulin-dependent diabetes mellitus (NIDDM), exhibits impaired glucose tolerance (IGT) at about 16 weeks of age. In this study, we investigated whether or not biotin, a water-soluble vitamin, improved the IGT of OLETF rats. To this end, we administered diets containing one of three levels of biotin, a high-biotin diet (BH), a normal-biotin diet (BN) and a basal-biotin diet (BB), to OLETF rats up to 24 weeks of age. An oral glucose tolerance test (OGTT) was performed four times between 13 and 22 weeks of age. The administration of a BH corrected the IGT of OLETF rats. Upon further investigation, we found that insulin secretion in the OLETF-BH rats was decreased to a significant extent, signaling that the hyperinsulinemia typical to the OLETF-BH rats had clearly improved. Body weights were significantly lower in the OLETF-BH group than in the other OLETF groups, even though the OLETF-BH rats showed a significantly higher average daily food intake. The body weight gain of the OLETF-BH rats followed the same tendency as the control-LETO (Long Evans Tokushima Otsuka) rats (LETO-BB and LETO-BN). These results demonstrate that a high-level biotin diet can improve the glucose handicap in NIDDM rats.

Effects of dietary biotin on enhanced sucrose intake and enhanced gustatory nerve responses to sucrose seen in diabetic OLETF rat.

We used the sucrose preference test and taste nerve recording to investigate the effect of dietary biotin on the abnormal sucrose taste sensitivity and preferences seen during the course of diabetes mellitus. For this, we used Otsuka Long-Evans Tokushima fatty (OLETF) rats. The chorda tympani nerve (CT nerve) response to sucrose (> 1 M) was of greater relative magnitude in OLETF rats than in non-diabetic control (Long-Evans Tokushima Lean, LETO) rats, but the responses to other basic taste stimuli (such as HCl, quinine-HCl and L-glutamic acid) did not differ between the two groups. In behavioral experiments using a two-bottle preference test, solution intake for sucrose (> 50 mM) was higher in OLETF rats than in LETO rats. The neural responses to sucrose (1.5-2 M) in OLETF rats were lower when given a biotin-high diet (BH-OLETF) than when given a biotin-basal diet (BB-OLETF), but this was not true of the other basic tastes. However, there were no significant differences between BH-OLETF and BB-OLETF rats in terms of sucrose solution intake. These findings suggest that the enhanced sugar sensitivity observed in OLETF rats is probably the result of a genetic difference between OLETF and LETO rats, though the discrepancy can be modified by the dietary biotin level.

Percutaneous absorption of biotin in healthy subjects and in atopic dermatitis patients.

The study was designed to test the ability of sequential applications of biotin-containing ointment to increase serum biotin levels. Twenty atopic dermatitis patients (mean age, 20.5 yr) and 11 healthy subjects (mean age, 25.5 yr) volunteered to participate in this study. The diagnosis of atopic dermatitis was established dermatologically. Seven grams per day of ointment containing 0.3% biotin and 1-4 g per day of steroid ointment were both applied sequentially. The healthy subjects applied only biotin ointment. The biotin concentration was determined microbiologically. Before biotin treatment, the average serum biotin level was significantly lower in atopic dermatitis patients than in healthy subjects. The percutaneous application of biotin-containing ointment caused a significant increase in the serum biotin concentration in both healthy subjects (from 41.5 +/- 10.0 to 50.2 +/- 9.2 nmol/L) and in atopic dermatitis patients (from 27.9 +/- 17.4 to 50.7 +/- 21.6 nmol/L), especially in patients whose initial level was low, and also could be effective in regulating the atopic allergic response involving eosinophils. In conclusion, biotin appears to be readily absorbed through both normal and dermatitis-affected human skin.

Newly developed model for vitamin K deficiency in germfree mice.

The mechanism of induction of vitamin K (VK) deficiency in newborn babies and antibiotics-treated patients has not entirely been clarified because of the difficulty in preparing the true VK deficient model-animals and the complication in an assay system for VK derivatives and of their metabolites until now. Germfree animal is thought to be an useful tool to establish a primary VK deficiency not caused by VK antagonists etc., because of the lack of their intestinal flora. Germfree (GF) and conventional (CV) ICR/JCL male mice, 12-13 week-old were used in this experiment. VK deficient (K-Def), menaquinone-4 (MK-4) supplemented (MK-4), and VK3 (menadione) supplemented diet (K3) were fed to the mice in both GF and CV states. After 8 days, severe VK deficient symptoms were occurred only in GF-K-Def group, whereas not at all in CV-K-Def group. Both prothrombin time (PT) and activated partial thromboplastin time (APTT) were also prolonged only in GF-K-Def group. From the HPLC analysis of MK-4 content in liver, it was suspected that the content of MK-4 which has been thought to be an active form of VK was not necessarily paralleled with the degree of VK deficiency.

Antibiotic-induced vitamin K deficiency and the role of the presence of intestinal flora.

Cephalosporin antibiotics with N-methyl-thio-tetrazole (NMTT) side chains have been known to be associated with the development of hypoprothrombinemia. However, it has not been fully established whether these symptoms are induced by an inhibition of vitamin K production by intestinal microorganisms or by an inhibitory action of these antibiotics on endogenous vitamin K metabolism. Therefore, an attempt has been made to clarify the above-mentioned ambiguity by using germfree mice in which primary vitamin K deficiency can be established within a short experimental period. Germfree (GF) and conventional (CV) ICR male mice, 8-13 weeks old were used in this experiment. Vitamin K deficient (Def) and menaquinone-4 supplemented diet (MK-4) were fed to the mice in both rearing conditions. In the antibiotic-treated group, sodium latamoxef (LMOX, 300 mg/kg B.W./day) was intraperitoneally administered once a day, and in the control group the same volume of saline (Saline) was administered. Severe vitamin K deficient symptoms were observed in the GF-K-Def-LMOX group, and both prothrombin time (PT) and activated-partial thromboplastin time (APTT) values were prolonged on the 8th day of the experimental period compared with the GF-K-Def-Saline group. Furthermore the mortality rate of GF-K-Def-LMOX group was comparatively higher than that of the Saline group. This study has provided evidence that vitamin K deficiency is amplified by an administration of LMOX even in the absence of intestinal flora.

Reduction in carbonic anhydrase activity in the tongue epithelium and submandibular gland in zinc-deficient rats.

We investigated the effects of zinc deficiency on carbonic anhydrase (CA) activity in the tongue epithelium and submandibular gland in rats. Male 4-week-old SD rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low-zinc), or 33.7 (zinc-sufficient) mg zinc/kg diet for 6 weeks. Rats in the fourth group (receiving 33.7 mg zinc/kg) were pair-fed against the zinc-deficient rats. Biochemical analysis at the end of the experimental period indicated that zinc deficiency significantly reduced CA activity in the tongue epithelium and submandibular gland, and the CA activity levels in these tissues seemed to parallel the dietary zinc levels. By enzyme histochemistry, an intensely positive reaction for CA was observed in the middle and basal regions of the taste buds in the circumvallate papilla in the zinc-sufficient and pair-fed (control) rats. The cells in von Ebner's glands also showed a strong positive reaction in control rats, although only a weak reaction product was found in zinc-deficient rats. These results suggest that CA activity is affected by the dietary content of zinc, which is considered to be an indispensable factor for the maintenance of normal taste sensation.

Biochemical consequences of biotin deficiency in osteogenic disorder shionogi rats.

The biological consequences of biotin deficiency in rats were investigated using osteogenic disorder Shionogi rats which have a hereditary defect in ascorbic acid synthesizing ability. Decrease of liver ascorbic acid content and fasting plasma glucose and an increase of plasma non-esterified fatty acid (NEFA) appeared in biotin deficient rats fed a diet containing 200 mg ascorbic acid per 100 g diet, compared with the pair fed control. On the other hand, in the case of rats fed a diet containing 500 mg ascorbic acid, although the clinical features of biotin deficiency developed, the ascorbic acid contents of liver and adrenal gland increased in comparison with those of AsA 200 mg groups, and the alterations of plasma levels of glucose and NEFA were improved partially in glucose and greatly in NEFA, respectively. This suggests that ascorbic acid may be consumed in the improvement of the metabolic impairments induced by biotin deficiency.

Urinary mercury monitoring of university staff and students occasionally exposed to mercury vapor.

To monitor the exposure to mercury (Hg) vapor among university staff members and students who occasionally handle elemental Hg in laboratory experiments, urine samples were collected at health examinations conducted by the Health Service Center, University of Tokyo, for six years. Geometric mean of urinary Hg concentrations of 343 samples collected from 234 subjects was 1.61 micrograms Hg/g creatinine (Cr), with the range of 0.30 to 9.31 micrograms Hg/g Cr. Elevated urinary Hg levels, i.e. 3 micrograms Hg/g Cr or higher, were found only among the subjects who worked in several laboratories. This urinary Hg level is judged to correspond to 1-2 micrograms/m3 of air Hg concentration in working areas. The contribution of dental amalgam fillings to urinary Hg excretion, though it exists, was concluded to be small from the result of multiple regression analysis.

Surgical procedure and results of repair of massive tears of the rotator cuff.

One hundred eighty-seven patients (189 shoulders) were treated surgically between 1970 and 1992 for massive rotator cuff tears using either a tendon-to-tendon repair or the McLaughlin procedure. The age of the patients ranged from 20 to 86 years; 95% of them were 45 years or older. The average followup was 6 years 9 months. Excellent or good functional results were attained in 93% of patients. Thirty-three percent of those who underwent tendon to tendon repair complained of pain after overuse compared with only 18% who had the McLaughlin Procedure.

Long-term zinc deficiency decreases taste sensitivity in rats.

The effects of zinc deficiency on taste sensitivity were examined in rats by recording the electrophysiological responses of the chorda tympani (CT) nerve and by use of a preference test. Male 4-wk-old Sprague-Dawley rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low zinc) or 33.7 (zinc-sufficient) mg zinc/kg diet. A fourth group was pair-fed the zinc-sufficient diet (with respect to the zinc-deficient rats). A two-bottle preference test using 0.15 mol/L NaCl and water revealed that NaCl preference was greater in the zinc-deficient and low zinc groups than in the control groups (zinc-sufficient and pair-fed) after 4 d of feeding. In the case of quinine hydrochloride solution (0.01 mmol/L), the preference was greater in zinc-deficient rats than in the other groups after 9 d, and the low zinc rats never showed a preference. Electrophysiological recording indicated that in the zinc-deficient rats, the CT nerve response to 0.20 mol/L NaCl was significantly less than that in the control rats after 21 d of feeding. In the low zinc rats, this response was significantly less than in the control rats after 35 d. The responses to quinine hydrochloride (0.02 mol/L), L-glutamic acid, HCl (0.01 mol/L) and NH(4)Cl (0.25 mol/L) in the zinc-deficient rats were not significantly reduced until d 42. These findings suggest that long-term zinc deficiency decreases taste sensitivity at the level of the CT nerve and that the change in NaCl preference due to zinc deficiency occurs before any change in NaCl taste sensitivity.