The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

SARC-F Validation and SARC-F+EBM Derivation in Musculoskeletal Disease: The SPSS-OK Study.

OBJECTIVES: To validate the SARC-F questionnaire for sarcopenia screening in musculoskeletal disease setting, and to assess improvements in diagnostic accuracy by adding "EBM" (elderly and body mass index information) to the SARC-F. DESIGN: Diagnostic accuracy study. SETTING AND PARTICIPANTS: The center involved in this study was located in an urban area of Kobe City, Japan. People with musculoskeletal disease in the knee, hip, or spine who were scheduled for surgical treatment were included. MEASUREMENTS: Sarcopenia was evaluated using the Asian Working Group for Sarcopenia (AWGS) and the European Working Group on Sarcopenia in Older People (EWGSOP2), which included bioimpedance, handgrip strength, and gait speed. Patients answered the SARC-F questionnaire and their body mass index was measured. SARC-F and "EBM" information were combined into an original score. The sensitivities, specificities, and areas under the receiver operating characteristic curve (AUC) were estimated and compared to identify sarcopenia. RESULTS: A total of 959 patients were included. Sarcopenia by AWGS criteria was identified in 36 (3.8%) patients. SARC-F had a sensitivity of 41.7% and specificity of 68.5%. SARC-F+EBM had a sensitivity of 77.8% and specificity of 69.6%, with substantial improvement in sensitivity (P<0.001). The AUCs for SARC-F and SARC-F+EBM were 0.557 (95% confidence interval [CI] 0.452-0.662) and 0.824 (95% CI 0.762-0.886), respectively (P<0.001). Similar results were obtained when EWGSOP2 criteria were used as the reference standard. CONCLUSION: The SARC-F alone is not adequate for finding cases in musculoskeletal disease settings. SARC-F+EBM significantly improved the sensitivity and overall diagnostic accuracy of the SARC-F for screening sarcopenia. SARC-F+EBM is potentially useful for screening sarcopenia in different ethnic and disease settings.

Measurement of delta-aminolevulinic acid synthetase activity in human erythroblasts.

A new, specific, and simple method for the determination of delta-aminolevulinic acid (ALA) synthetase activity in human bone marrow cells has been developed. ALA synthetase of erythroblasts was partially purified so as to permit the use of [(14)C]succinyl-CoA as a substrate for this enzyme. In this enzyme preparation there were negligible activities of succinyl-CoA hydrolase, alpha-ketoglutarate dehydrogenase, and succinyl-CoA synthetase and there was no activity of ALA dehydrase. The ALA formed from [(14)C]succinyl-CoA has been isolated by column chromatography. Radioactivity in the eluate from the column has been proved by paper chromatography to be exclusively that of [(14)C]ALA. The entire assay can be completed within 4 h, and [(14)C]succinyl-CoA was incorporated into [(14)C]ALA on the order of several percent. Moderate to marked decreases of ALA synthetase activity have been demonstrated in the erythroblasts of all cases of sideroblastic anemia. In the cases of iron deficiency anemia, on the other hand, normal or slightly elevated activity has been obtained.

Accumulation of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole and 2-aminodipyrido[1,2-a:3',2'-d]imidazole, carcinogenic glutamic acid pyrolysis products, in plasma of patients with uremia.

In order to investigate the exposure of humans to 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole [(Glu-P-1) Chemical Abstracts Service:67730-11-4] and 2-aminodipyrido[1,2-a:3',2'-d]imidazole [(Glu-P-2) Chemical Abstracts Service:67730-10-3], carcinogenic heterocyclic amines, we developed a high-performance liquid chromatography method to detect Glu-P-1 and Glu-P-2 in biological samples, and compared the plasma levels of the carcinogens in normal subjects with those in uremic patients in which higher incidence of malignancy has been reported. Glu-P-1 and Glu-P-2 levels in plasma of uremic patients before induction of hemodialysis treatment were 12.62 +/- 3.65 (SD) pmol/ml (n = 5) and 14.81 +/- 5.17 pmol/ml (n = 5), respectively, whereas Glu-P-1 and/or Glu-P-2 could be detected in only two of seven normal subjects and the levels were lower than 3.1 pmol/ml. Approximately 10% of these carcinogens in plasma of uremic patients could be removed by the first hemodialysis treatment, and reasonable amounts of these carcinogens could be detected in the dialysate of uremic patients. However, significant amounts of Glu-P-1 and Glu-P-2 were still detected in plasma of all uremic patients even after 1 month-hemodialysis treatments. These results suggest that one of the excretory pathways of these carcinogens is via kidney.

Joint awareness after total knee arthroplasty is affected by pain and quadriceps strength.

INTRODUCTION: There is a growing interest in the use of patient-reported outcomes to provide a more patient-centered view on treatment. Forgetting the artificial joint can be regarded as the goal in joint arthroplasty. The goals of the study were to describe changes in joint awareness in the artificial joint after total knee arthroplasty (TKA), and to determine which factors among pain, knee range of motion (ROM), quadriceps strength, and functional ability affect joint awareness after TKA. HYPOTHESIS: Patients undergoing TKA demonstrate changes in joint awareness and joint awareness is associated with pain, knee ROM, quadriceps strength, and functional ability. PATIENTS AND METHODS: This prospective cohort study comprised 63 individuals undergoing TKA, evaluated at 1, 6, and 12 months postoperatively. Outcomes included joint awareness assessed using the Forgotten Joint Score (FJS), pain score, knee ROM, quadriceps strength, and functional ability. RESULTS: Fifty-eight individuals completed all postoperative assessments. All measures except for knee extension ROM improved from 1 to 6 months. However, there were no differences in any measures from 6 to 12 months. FJS was affected most greatly by pain at 1 month and by quadriceps strength at 6 and 12 months. DISCUSSION: Patients following TKA demonstrate improvements in joint awareness and function within 6 months after surgery, but reach a plateau from 6 to 12 months. Quadriceps strength could contribute to this plateau of joint awareness. LEVEL OF EVIDENCE: Prospective cohort study, IV.

Age-associated changes in rat plasma lipids, platelet fatty acids and prostacyclin release.

The effect of age on plasma lipids, platelet fatty acids and prostacyclin release was studied in the rat. The contents of arachidonic acid, cholesterol, bile acids and free fatty acids in the plasma of aged rats (15 months old) were higher than those of young rats (3 months old). No significant differences in fatty acid composition of platelet lipids and release of prostacyclin from aortas between young and aged rats were observed. The data suggest that plasma lipids may play a more important role in the development of cardiovascular disease with increasing age than prostaglandins do.

Effect of age on the modification of rat plasma lipids by fish and soybean oil diets.

The effects of sardine and soybean oils on plasma lipids have been studied in young and aged rats. Plasma cholesterol and bile acids of aged rats fed on a sardine oil diet decreased to a greater degree than those of young rats. Cholesterol, bile acids and phospholipids of the soybean oil diet group decreased only in aged rats. Increases in plasma eicosapentaenoic (sardine) and linoleic (soybean) acid levels of aged rats were observed to be greater than those of young rats. These results indicate that the age enhances the effects of fish and soybean oils on plasma lipids by suppressing their characteristic fatty acid metabolism.

Triphenyltin fluoride (TPTF) as a diabetogenic agent. TPTF induces diabetic lipemia by inhibiting insulin secretion from morphologically intact rabbit B-cell.

Recent work in our laboratory has shown that oral administration of triphenyltin fluoride (TPTF) evokes hypertriglyceridemia in rabbits. The present experiments were conducted to elucidate the mechanism of TPTF-induced hypertriglyceridemia in rabbits by a combined biochemical and ultrastructural approach. After a single TPTF administration, fasting blood glucose and plasma triglyceride levels increased significantly (P less than 0.02) for about 20 days. On the other hand, both plasma and adipose tissue lipoprotein lipase (LPL) activity was markedly decreased (P less than 0.001) during this period, and triglyceride production rates on day 2 after TPTF administration was significantly decreased (P less than 0.01). Density-gradient ultracentrifugation showed a remarkable accumulation of chylomicron and VLDL in the composition of plasma lipoproteins. Insulin injection to the hypertriglyceridemic rabbits induced a significant recovery of the decreased plasma LPL activity with a concomitant decrease of plasma triglyceride levels, while abeyance of insulin injection resulted in a decrease of LPL activity again. A significant inhibition of insulin release in response to the loading of glucose, glucagon, or arginine was observed in the TPTF rabbits (P less than 0.02). Inhibition of glucagon release was also observed in the arginine-loading test (P less than 0.01). Electron microscopic studies showed small abnormalities in the pancreatic islets of TPTF-treated rabbits. These findings suggest that TPTF inhibits insulin release from rabbit islets, subsequently inducing diabetic lipemia due to the insulin deficiency. Furthermore, it is possible to provide a new animal model for diabetes and diabetic lipemia by administration of TPTF to rabbits.

Triphenyltin chloride inhibits superoxide production by human neutrophils stimulated with a surface active agent.

Treatment of human neutrophils with triphenyltin chloride (TPTCl)-inhibited superoxide (O-2) production stimulated with phorbol myristate acetate (PMA). TPTCl was more potent as inhibitor of O-2 production than other phenyltin compounds. The O-2 production by the xanthine oxidase-acetaldehyde system was not inhibited by TPTCl. This finding indicates that TPTCl does not itself react with O-2. Furthermore, TPTCl did not influence the isolated NADPH oxidase at all, though O-2 production of neutrophils stimulated with PMA in the presence of TPTCl was inhibited. These results indicate that TPTCl inhibits the activation process of the O-2 generating system.

Miconazole inhibition of platelet aggregation by inhibiting cyclooxygenase.

Platelet dysfunction was found in rabbits to which a dose of miconazole nitrate (1.6 mg/kg body wt) therapeutic for human subjects had been given intravenously. The present experiments were conducted to elucidate the mechanism of inhibitory effects of miconazole on platelet function. After administration of a single dose of miconazole, rabbit platelet aggregation induced by collagen and sodium arachidonate was inhibited significantly for approximately 24 hr. On the other hand, hypertriglycemia, one of the major side effects of this drug, was not seen during 2 days of observations, nor were any other outstanding manifestations observed. In in vitro experiments, miconazole nitrate (10 microM) also significantly inhibited rabbit and human platelet aggregation (P less than 0.01). Biochemical analyses revealed that the stimulant-induced formation of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2), metabolites via cyclooxygenase, was inhibited by miconazole nitrate in both human and rabbit platelets in vitro. PGE2 production was decreased dose-dependently with the increase of miconazole concentration (10 to 100 microM), and the decrease was in parallel with a decrease of TXB2 production. In addition, malondialdehyde (MDA) production of human and rabbit platelets induced by exogenous arachidonate and collagen was also inhibited significantly by miconazole. Chromatographic studies showed that the amount of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), a metabolite via lipoxygenase, was increased markedly in accordance with the miconazole-induced decrease of TXB2 and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) formation in both human and rabbit platelets. These results indicate that miconazole nitrate inhibits platelet cyclooxygenase, without affecting the stimulant-induced release of arachidonic acid from platelet phospholipids. Use of this drug in the treatment of systemic fungal infection appears to be increasing. Careful attention should be paid to the inhibitory effects of miconazole on platelet function, especially in the case of intravenous treatment.

Triphenyltin fluoride in vitro inhibition of rabbit platelet collagen-induced aggregation and ATP secretion and blockade of arachidonic acid mobilization from membrane phospholipids.

Recent studies have demonstrated that triphenyltin fluoride (TPTF) inhibits collagen-induced aggregation and ATP secretion of rabbit platelets in vivo [S. Manabe and O. Wada, J. Toxic. Sci. 6, 236 (1981)]. The aim of the present investigation was to test the effects in vitro of TPTF on platelet aggregation and to elucidate the mechanism of the inhibitory action by studying the release and metabolism of arachidonic acid and the cyclic AMP contents of rabbit platelets treated in vitro with TPTF. Although no inhibitory effect of TPTF was found on sodium arachidonate-induced platelet aggregation and ATP secretion, TPTF inhibited both reactions induced by collagen. Triphenylarsine and triphenylantimony did not inhibit, even at a concentration of 10(-3) M. The anti-aggregating concentration (IC50) of TPTF was 6.0 x 10(-6) M against collagen. TPTF had no inhibitory effect on the conversion of exogenous arachidonic acid to malondialdehyde (MDA) by platelets, while the collagen-induced production of arachidonate metabolites [MDA, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2] was remarkably inhibited by TPTF. Furthermore, TPTF apparently inhibited the collagen-induced release of arachidonic acid from platelets, although the formation of phosphatidic acid was not inhibited. Total cyclic AMP content after TPTF exposure was not changed significantly. These results indicate that TPTF inhibited the collagen-induced arachidonic acid release from platelet phospholipids, presumably by acting on phospholipase A2. Furthermore, it seems unlikely that the inhibition of arachidonic acid release by TPTF can be explained by the level of cyclic AMP in platelets.

The association of smoking and drinking habits with serum pepsinogens.

BACKGROUND: The influence of smoking on serum pepsinogen I has been assessed. However, still to be assessed are the influences of smoking on pepsinogen II and drinking on serum pepsinogens. METHODS: Data were collected from 13,381 employees by questionnaire and serum tests. Multiple regression analyses were done with logarithms of serum pepsinogen I (LPI), pepsinogen II (LPII) or pepsinogen I/II ratio (LI/II) as a criterion variable and as categorized explanatory variables, sex, age, subjective symptoms in the stomach, past history of peptic ulcer, current smoking dose, past smoking amount, drinking habit and current drinking dose. RESULTS: Current smoking dose showed dose-dependent positive associations with LPI and LI/II: Past smoking amount yielded weakly dose-dependent negative associations with LPI and LI/II: Current drinking dose showed dose-dependent negative associations with LPI and LPII. CONCLUSION: Current smoking elevates pepsinogen I and the I/II ratio, and it may be necessary to consider the effect of smoking when pepsinogens are used as markers for gastric cancer. Drinking reduced pepsinogen I and II, but the effect was not so large.

Suppression of GABA-induced chloride current by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2).

The effect of a convulsive agent, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), on the GABA-induced chloride current (ICl) in dissociated mouse sensory neurons was investigated using the whole cell clamp method. Trp-P-2 reversibly suppressed the GABA-induced ICl in a dose-dependent manner. The IC50 for Trp-P-2 on the ICl evoked by 3 microM GABA was 11.1 microM. Ro15-1788 had no effect on the suppressive action of Trp-P-2. These results suggest that the observed effect of Trp-P-2 is mediated by its action as an antagonist at GABAA receptors.

Identification of carcinogenic tryptophan pyrolysis products in human bile by high-performance liquid chromatography.

The carcinogenic tryptophan pyrolysis products 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) have been demonstrated to be present in human bile through use of a high-performance liquid chromatography (HPLC) method. The method consists of the acid-induced release of the carcinogens from bile components and their extraction with methylene chloride and subsequent quantification by HPLC. In seven subjects who had received catheterization of the bile duct and external biliarydrainage, the average amounts of Trp-P-1 and Trp-P-2 excreted daily in the bile were 408 fmol/day (n = 7) and 864 fmol/day (n = 7), respectively. In one subject, furthermore, significant daily changes of these carcinogen levels in bile and plasma were confirmed during 2 weeks of observation. These results indicate that one of the excretory pathways of these carcinogens is via bile. Our data also may suggest that Trp-P-1 and Trp-P-2 are derived from everyday foods.

Exposure level monitor of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, a dietary carcinogen, in rabbits.

The present investigation describes a method for the detection of minute amounts of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), a carcinogenic tryptophan pyrolysate, bound to the hemoglobin of erythrocytes and plasma from rabbits dosed orally with the dietary carcinogen. The method consists of the acid-induced release of the dietary carcinogen adducts as the free carcinogen and their extraction with methylene chloride and subsequent quantitation by high-performance liquid chromatography (HPLC). With this method, Trp-P-1 levels in plasma, platelets, and red blood cells were monitored for 12 weeks to determine a suitable indicator for estimating the exposure levels of the dietary carcinogen. The results suggest that Trp-P-1 in red blood cells that bound covalently to the hemoglobin is the most suitable for monitoring the long-term exposure levels.

Detection of carcinogenic glutamic acid pyrolysis products in Worcestershire sauce by high-performance liquid chromatography.

Commercially available Worcestershire sauce was analyzed for mutagenic and carcinogenic glutamic acid pyrolysis products using high-performance liquid chromatography. These carcinogenic heterocyclic amines were found to be present in all brands of Worcestershire sauce analyzed. The identity of the carcinogens was confirmed by spectrometric analyses and the SOS umu-test. The concentrations of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) in the Worcestershire sauce were 695 +/- 329 pmol/liter (mean +/- SD, n = 5) and 1,839 +/- 1,321 pmol/liter (n = 5), respectively.

Increased apoptosis in a variety of tissues of zinc-deficient rats.

Zinc deficient rats were prepared to investigate histopathological changes in thymus, testis, skin, esophagus, kidney and liver and the relationship between these changes and apoptosis. Seven-week-old male SD rats were given a Zn deficient diet (0% Zn diet) or a standard diet (0.02% Zn diet). The above-mentioned organs were excised 1, 2, 3, 4, 5, 10, 13, and 34 weeks after initiating diet administration. Then, these organs were examined morphologically, and apoptotic changes were analyzed by either the TdT- mediated dUTP - biotin nick end labeling (TUNEL) or electrophoresis. Significant morphological changes were seen only in rats on the 0% Zn diet. After 4 weeks, atrophy of the thymus was seen. After 5 weeks, oligospemia was observed, and after 10 weeks, testicular atrophy accompanied by the loss of sperm cells and spermatocytes was confirmed. In addition, after 10 weeks, thickening of epithelia was seen in the skin and esophagus of rats on the 0% diet. During the observation period, no marked morphological changes were observed in the liver or kidney. In the thymus and testis of rats on the 0% Zn diet, prior to detecting any morphological changes, increases in apoptosis were confirmed at 1 and 3 weeks after initiating diet administration, respectively. In the kidney and liver, TUNEL positive cells appeared after 13 and 34 weeks, respectively. These observations suggest that the functional and morphological changes in the thymus and testis of rats on the 0% Zn diet are caused by increased apoptosis, and that even when the supply of Zn is terminated for only a short period of time, immunocytes and germ cells can not survive or regenerate sufficiently. Again, the fact that even in the liver and kidney, apoptosis was observed when administration of the 0% Zn diet was prolonged suggests that the appearance of apoptosis is dependent on the amount of Zn in tissues. In addition, the fact that increases in apoptosis were confirmed in the skin of rats on the 0% Zn diet, but not in the esophagus of these rats suggests that apoptosis does not directly cause thickening of stratified squamous epithelium in Zn deficient rats.

Lithium deficiency reduces thymus weight and alters differential blood count in rats.

Effects of lithium (Li) deficiency and/or immobilization stress on the thymus weight and differential blood count of rats were studied. The thymus weight of the rats fed a low Li diet were lighter than those of rats fed a Li supplemented diet, whether the rats were exposed to stress or not. Of the rats not exposed to stress, those in the low Li diet group showed a significant decrease in the ratios of neutrophils and T helper lymphocytes. However, there was an increase in the total number of lymphocytes in the low Li dietary group. It was shown that Li deficiency altered the responses to stress in rats.

Distribution and chromium-binding capacity of a low-molecular-weight, chromium-binding substance in mice.

The distribution of low-molecular-weight, chromium-binding substance (LMWCr) and high-molecular-weight, chromium-binding substance (HMWCr) in the organ cytosol were analyzed by means of Sephadex G-25 gel filtration, after a single i.p. injection of K2Cr2O7 (280 mumol, Cr/Kg) to mice (male dd, 23 +/- 2 g). The amount of Cr in LMWCr per mouse was highest in the liver (83 micrograms), followed by those in the kidney (10 micrograms) and other organs (3-1 micrograms), with lesser amounts of Cr in HMWCr in all the organs. In these organs LMWCr was found to bind 3-28 times the amount of Cr to that in the in vivo binding after the in vitro incubation with K2Cr2O7 at 37 degrees C, showing a high Cr binding capacity of the substance. No inductive formation of LMWCr was observed in the liver even after daily repetitive administration of Cr (150 mumol/Kg, 4 days). Time course studies on the liver and the kidney of mice injected with K2Cr2O7 showed no difference in the accumulation of Cr in LMWCr and in the ratio of Cr in LMWCr to that in HMWCr between the organs at intervals of from 5 min to 24 hr after the injection. The comparative affinity of Cr(III) for LMWCr and for the serum proteins decreases in the order LMWCr, transferrin, albumin. The transfer of Cr from LMWCr to albumin and vice versa was almost negligible. However, significant amounts of the metal transfer was found from LMWCr to transferrin and vice versa, and from albumin to transferrin. These findings suggest that LMWCr is distributed widely in the body and it quickly binds invaded Cr in stable form at an organ site, especially in the liver, with participation of albumin or/then transferrin. This supports the hypothesis that LMWCr plays a large role in Cr detoxification.