Does weight-bearing versus non-weight-bearing pain reflect different pain mechanisms in knee osteoarthritis?: the Multicenter Osteoarthritis Study (MOST).

OBJECTIVE: Knee osteoarthritis (OA) is predominantly characterized by pain with weight-bearing activities. Pain at rest also occurs but the mechanisms for this are not clear. We evaluated the relations of nociceptive signal alterations to weight-bearing and non-weight-bearing pain in knee OA. DESIGN: We used data from a NIH-funded longitudinal cohort of older adults with or at risk of knee OA. We evaluated quantitative sensory testing (QST) measures (pressure pain threshold (PPT) at patellae and the wrist; mechanical temporal summation (TS); conditioned pain modulation (CPM)). Each WOMAC pain question was dichotomized as having at least moderate pain, and we further categorized them as weight-bearing pain and non-weight-bearing pain. We evaluated the relation of QST measures to each pain outcome using logistic regression, adjusting for potential confounders. RESULTS: 2,749 participants (5,479 knees) were included (mean age 64 ± 11, 57% female). Each SD unit decrease in patellar PPT was associated with greater odds of both weight-bearing pain (OR 1.51 (95% CI 1.27, 1.79)) and non-weight-bearing pain (OR 1.46 (1.20-1.77)), while wrist PPT was associated with greater odds of weight-bearing pain (OR 1.27 (1.15, 1.39)) but only with pain during sitting/lying (OR 1.20 (1.01, 1.43)). TS was significantly associated with greater odds of pain with walking and stairs (OR 1.11 (1.01, 1.23), 1.11 (1.03, 1.20), respectively). CPM was not associated with any pain outcomes. CONCLUSIONS: Our findings challenge the hypothesis that non-weight-bearing pain may reflect greater pain sensitization and/or inefficient CPM than weight-bearing pain in knee OA, suggesting other mechanisms are likely responsible.

Accuracy of spinal curvature assessed by a computer-assisted device and anthropometric indicators in discriminating vertebral fractures among individuals with back pain.

UNLABELLED: This study examined the accuracy of thoracic and lumbar kyphotic angles as well as anthropometric indicators for discriminating patients with vertebral fracture among Japanese women >50 years old with back pain. Along with region-specific kyphotic angles and anthropometric indicators, the combination of thoracic and lumbar kyphotic angles offered the highest accuracy. INTRODUCTION: Vertebral fractures have been associated with thoracic kyphosis. However, reports on lumbar kyphotic changes in association with vertebral fracture are scarce. This study investigated the accuracy of thoracic kyphotic angle (TKA) and lumbar kyphotic angle (LKA) measurements as well as anthropometric indicators (wall-occiput distance (WOD) and rib-pelvis distance (RPD)) in discriminating patients with vertebral fracture. METHODS: Lateral radiographs of the spine were obtained in 70 postmenopausal Japanese women who visited an orthopedic clinic with low back pain (mean age, 76.2 ± 9.0 years). Radiographic vertebral fracture was diagnosed using quantitative measurement according to Japanese criteria. Osteoarthritis (OA) was defined as Kellgren-Lawrence (KL) grade 3 or higher. TKA and LKA were measured using SpinalMouse®. WOD and RPD were also measured. RESULTS: At least one vertebral fracture was present in 49 subjects (70 %). Women with vertebral fractures showed significant increases in LKA, TKA + LKA, and WOD and decreases in RPD. Logistic regression analysis showed significant association between TKA + LKA and vertebral fracture independent of the presence of OA. Receiver operating characteristic analysis revealed that TKA was useful for discriminating thoracic fractures (area under the curve (AUC), 0.730) and LKA was useful for lumbar fractures (AUC, 0.691). The combination of TKA + LKA offered the highest accuracy for detecting thoracic, lumbar, and any vertebral fractures, with AUCs of 0.779, 0.728, and 0.783, respectively. WOD and RPD showed low-to-moderate accuracies for thoracic, lumbar, and any vertebral fractures. CONCLUSIONS: Assessment of spinal kyphosis by SpinalMouse® as well as anthropometric indicators proved useful in discriminating subjects with vertebral fractures. These convenient and radiation-free methods could contribute to early diagnosis of vertebral fractures and subsequent appropriate treatment, thus preventing additional osteoporotic fractures.

Core level excitations--a fingerprint of structural and electronic properties of epitaxial silicene.

From the analysis of high-resolution Si 2p photoelectron and near-edge x-ray absorption fine structure (NEXAFS) spectra, we show that core level excitations of epitaxial silicene on ZrB2(0001) thin films are characteristically different from those of sp(3)-hybridized silicon. In particular, it is revealed that the lower Si 2p binding energies and the low onset in the NEXAFS spectra as well as the occurrence of satellite features in the core level spectra are attributed to the screening by low-energy valence electrons and interband transitions between π bands, respectively. The analysis of observed Si 2p intensities related to chemically distinct Si atoms indicates the presence of at least one previously unidentified component. The presence of this component suggests that the observation of stress-related stripe domains in scanning tunnelling microscopy images is intrinsically linked to the relaxation of Si atoms away from energetically unfavourable positions.

Associations of vertebral deformities and osteoarthritis with back pain among Japanese women: the Hizen-Oshima study.

UNLABELLED: We examined the spinal distribution of the types of vertebral deformities and the associations of vertebral deformities and osteoarthritis with back pain in Japanese women. Midthoracic and upper lumbar vertebrae were more susceptible to deformity. Vertebral deformity and osteoarthritis were frequent and were associated with back pain. INTRODUCTION: Vertebral fractures due to osteoporosis and osteoarthritis are both common and significant health problems in aged people. However, little is known about the descriptive epidemiology of the individual deformity types and the relative clinical impact in women in Japan. METHODS: Lateral radiographs were obtained from 584 Japanese women ages 40 to 89 years old. Deformities were defined as vertebral heights of more than 3 standard deviations (SDs) below the normal mean. Osteoarthritis was defined as Kellgren-Lawrence (KL) grade 2 or higher. Information on upper or low back pain during the previous month was collected by questionnaire. We compared the spinal distribution of the three types of vertebral deformities (wedge, endplate, and crush) typical of fractures and examined the associations of number and type of vertebral deformities and osteoarthritis with back pain. RESULTS: Fifteen percent of women had at least one vertebral deformity and 74% had vertebral osteoarthritis. The prevalence of upper or low back pain was 30.1%. Deformities were most common in the midthoracic and upper lumbar regions and wedge was the frequent type, followed by endplate and crush. Multiple logistic regression analysis showed that the odds of back pain was 3.0 (95% CI 1.5-6.3) times higher for women with a single wedge deformity and 3.2 (95% CI 1.0--0.6) times higher for women with two or more wedge deformities, compared to women with no wedge deformity. Vertebral osteoarthritis was associated with back pain (OR 1.8, 95% CI 1.1-2.9), independent of other covariates including age and deformities. CONCLUSION: Our results in this group of Japanese women are similar to and consistent with results reported previously in other populations of Japanese and Caucasians.

Beneficial effect of risedronate for preventing recurrent hip fracture in the elderly Japanese women.

SUMMARY: A 36-month observational study compared the incidence of unaffected side hip fracture in Japanese female osteoporosis patients with a history of hip fracture between 173 patients receiving risedronate and 356 risedronate-untreated controls. New hip fractures were significantly less frequent in the risedronate group, suggesting a preventive effect in high-risk patients. INTRODUCTION: The purpose of this study was to investigate the preventive effect of risedronate on second hip fracture immediately following a first hip fracture in Japanese female osteoporosis patients with unilateral hip fracture. METHODS: We conducted a prospective matched cohort study in 184 patients treated with risedronate and 445 patients not receiving risedronate after discharge from hospital. Both groups were followed-up for 36 months, and the incidence of unaffected side hip fracture and the frequency of adverse events were assessed. RESULTS: Efficacy could be investigated in 173 patients from the risedronate group and 356 patients from the control group. Hip fracture was detected in 5 and 32 patients, respectively. Kaplan-Meier estimates of the 36-month fracture incidence were 4.3% in the risedronate group and 13.1% in the control group (P = 0.010, log-rank test). The hazard ratios (95% confidence intervals) obtained by univariate and multivariate analysis were 0.310 (0.121-0.796) and 0.218 (0.074-0.639), respectively, indicating a significantly lower incidence of unaffected side hip fracture in the risedronate group. Adverse events occurred in 38 patients (48 events) from the risedronate group and 94 patients (108 events) from the control group, with serious adverse events in 21 patients (26 events) and 78 patients (88 events), respectively. CONCLUSIONS: No significant differences were observed between the two groups. The incidence of unaffected side hip fracture was significantly lower in the risedronate group. Accordingly, risedronate may have a preventive effect on hip fracture in high-risk Japanese female osteoporosis patients for fracture with a history of unilateral hip fracture.

Bone mineral densities in patients with developmental dysplasia of the hip.

UNLABELLED: Bone mineral density (BMD) of the lumbar spine, ultradistal radius, and calcaneus were significantly higher in the developmental dysplasia of the hip (DDH) patients than in the controls. Therefore, our data suggest that BMDs at different skeletal sites are greater in patients with DDH than in healthy women. INTRODUCTION: DDH has been acknowledged as a potentially preosteoarthritic condition that results in the development of hip osteoarthritis. Patients with DDH have been reported to have abnormal morphology of the pelvis and spine. Additional research, including that of bone quality, needs to be conducted to elucidate the pathogenetic mechanism of this disease. We therefore sought to determine whether BMD differs between healthy women and women with DDH. METHODS: We measured BMD in 40 women who were scheduled to undergo pelvic osteotomy for DDH (average age, 45.3 years) and in 31 healthy women used as age-matched controls (average age, 47.5 years). BMDs of the lumbar spine, radius, and calcaneus were measured. RESULTS: BMDs of the lumbar spine, ultradistal radius, and calcaneus were significantly higher in the DDH patients than in the controls. CONCLUSIONS: Therefore, our data suggest that BMDs at different skeletal sites are greater in patients with DDH than in healthy women.

Quantitative ultrasound and radiographic absorptiometry are associated with vertebral deformity in Japanese Women: the Hizen-Oshima study.

UNLABELLED: We evaluated the ability of heel quantitative ultrasound (QUS) and metacarpal radiographic absorptiometry (RA) to identify subjects with vertebral deformities in Japanese women aged ≥40. Both QUS and RA were associated with vertebral deformities, and the estimated prevalence at each T-score widely varied with age. INTRODUCTION: Heel QUS and metacarpal RA have been used for screening patients to evaluate risk of osteoporotic fractures. The aim of this study was to evaluate the ability of QUS and RA to identify women with vertebral deformities in 570 Japanese women aged ≥40, and to estimate the prevalence of vertebral deformity at each T-score. METHODS: Calcaneal QUS and metacarpal RA were performed. Radiographic vertebral deformities were assessed by quantitative morphometry, defined as vertebral heights more than 3 SD below the normal mean. RESULTS: The receiver operating characteristic analysis showed that both calcaneal stiffness index (SI) and metacarpal bone mineral density (BMD) were associated with vertebral deformities. Using the T-score of -2.5 as a cutoff value, the specificity and sensitivity for identifying individuals with vertebral deformities was 65% and 83% for calcaneal SI, and 40% and 88% for metacarpal BMD, respectively. The prevalence of vertebral deformity was estimated using age-adjusted logistic regression models. Women with calcaneal SI T-score of -2.5 had a 2% estimated probability of vertebral deformity at age 40, and 22% at age 80. For metacarpal BMD T-score of -2.5, estimated probability was less than 1% at age 40, and 27% at age 80. CONCLUSION: Both calcaneal SI and metacarpal BMD were associated with prevalence of vertebral deformity. Furthermore, the prevalence widely varied with age at any given bone value.

Contribution of anti-CCP antibodies, proximal interphalangeal joint involvement, HLA-DRB1 shared epitope, and PADI4 as risk factors for the development of rheumatoid arthritis in palindromic rheumatism.

OBJECTIVES: To determine which variables at baseline are predictive for the development of rheumatoid arthritis (RA) from palindromic rheumatism (PR) in a Japanese population. METHODS: Anti-cyclic citrullinated peptide (anti-CCP) antibodies, joint involvement pattern, genotypes of HLA-DRB1, peptidylarginine deiminase (PADI4), and protein tyrosine phosphatase (PTPN22) were examined in 28 patients with PR at baseline, and their clinical outcome was prospectively evaluated. The same variables were also investigated in 38 healthy controls. RESULTS: Eleven out of 28 patients with PR developed RA. The prevalence of anti-CCP antibodies in the PR patients who developed RA was significantly higher compared to the patients who did not. Proximal interphalangeal (PIP) joint involvement at baseline was also predictive towards the development of RA. Compared with the controls, differences in the frequency of single-nucleotide polymorphism (SNP) on padi4_104 [T(RA susceptible)-->C(RA non-susceptible)] and the presence of an RA susceptible homozygote of the PADI4 haplotype were detected in patients with PR whereas we could not find any further difference in PR patients who developed RA compared to PR patients who do not develop RA in PADI4. None of the subjects possessed the PTPN22 SNP (1858C-->T). Cox regression analysis revealed that anti-CCP antibodies as well as PIP involvement are the most relevant variables for the development of RA from PR. None of the PR patients with either HLA-DRB1*SE alleles (or the HLA-DRB1*0405 allele) or anti-CCP antibodies developed RA. CONCLUSIONS: Anti-CCP antibodies, in relation to HLA-DRB1*SE carriership, and PIP involvement are predictive for the development of RA from PR in the Japanese population.

Cross talk between hedgehog and epithelial-mesenchymal transition pathways in gastric pit cells and in diffuse-type gastric cancers.

We previously reported hedgehog (Hh) signal activation in the mucus-secreting pit cell of the stomach and in diffuse-type gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is known to be involved in tumour malignancy. However, little is known about whether and how both signallings cooperatively act in diffuse-type GC. By microarray and reverse transcription-PCR, we investigated the expression of those Hh and EMT signalling molecules in pit cells and in diffuse-type GCs. How both signallings act cooperatively in those cells was also investigated by the treatment of an Hh-signal inhibitor and siRNAs of Hh and EMT transcriptional key regulator genes on a mouse primary culture and on human GC cell lines. Pit cells and diffuse-type GCs co-expressed many Hh and EMT signalling genes. Mesenchymal-related genes (WNT5A, CDH2, PDGFRB, EDNRA, ROBO1, ROR2, and MEF2C) were found to be activated by an EMT regulator, SIP1/ZFHX1B/ZEB2, which was a target of a primary transcriptional regulator GLI1 in Hh signal. Furthermore, we identified two cancer-specific Hh targets, ELK1 and MSX2, which have an essential role in GC cell growth. These findings suggest that the gastric pit cell exhibits mesenchymal-like gene expression, and that diffuse-type GC maintains expression through the Hh-EMT pathway. Our proposed extensive Hh-EMT signal pathway has the potential to an understanding of diffuse-type GC and to the development of new drugs.

Association of unipedal standing time and bone mineral density in community-dwelling Japanese women.

UNLABELLED: Bone mineral density (BMD) and physical performance of the lower extremities decrease with age. In community-dwelling Japanese women, unipedal standing time, timed up and go test, and age are associated with BMD while in women aged 70 years and over, unipedal standing time is associated with BMD. INTRODUCTION: The aim of this study was to clarify whether unipedal standing time is significantly associated with BMD in community-dwelling women. METHODS: The subjects were 90 community-dwelling Japanese women aged 54.7 years. BMD of the second metacarpal bone was measured by computed X-ray densitometry. We measured unipedal standing time as well as timed up and go test to assess physical performance of the lower extremities. RESULTS: Unipedal standing time decreased with increased age. Timed up and go test significantly correlated with age. Low BMD was significantly associated with old age, short unipedal standing time, and long timed up and go test. Stepwise regression analysis revealed that age, unipedal standing time, and timed up and go test were significant factors associated with BMD. In 21 participants aged 70 years and over, body weight and unipedal standing time, but not age, were significantly associated with BMD. CONCLUSION: BMD and physical performance of the lower extremities decrease with older age. Unipedal standing time, timed up and go test, and age are associated with BMD in community-dwelling Japanese women. In women aged 70 years and over, unipedal standing time is significantly associated with BMD.

Career choice and attitudes towards dental education amongst dental students in Japan and Sweden.

The purpose of this study was to identify and compare the perspectives of dental students towards their career choice and dental education in Japan and Sweden. One hundred and fourteen dental students from the Nippon Dental University, Japan and 43 dental students from the Karolinska Institutet, Sweden participated in this study. Information was derived from a self-answered questionnaire consisting of five items for career choice and six items for dental education. Chi-square test and Wilcoxon signed-rank test were used for comparison. Significant differences were detected for 10 questionnaire items between the two countries. Regarding motivation towards the career choice, 44% of Swedish students indicated interpersonal motives related to helping other people, whereas 32% of Japanese students indicated expectations of their family in the dental profession. As future career options, 64% of Japanese and 47% of Swedish students planned to work as general dentists. More Swedish students (37%) preferred specialisation than Japanese students (17%). Nearly three-quarters of the Swedish students were satisfied with the teaching faculty of their school, whilst only 32% of the Japanese students indicated content. The perspectives of dental students were different in Japan and Sweden. This study provides a description of the perspectives of Japanese and Swedish dental students and enables better understanding of career decision and dental curriculum issues.

GASDERMIN, suppressed frequently in gastric cancer, is a target of LMO1 in TGF-beta-dependent apoptotic signalling.

Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

Kazakhstan: a folate-deficient area?

OBJECTIVE: In the Republic of Kazakhstan, located in central Asia, the traditional diet consists mainly of meat, and vegetable intake tends to be deficient. This eating lifestyle may contribute to folate deficiency, which is closely linked to abnormal homocysteine (HCY) metabolism. SUBJECTS AND METHODS: In order to evaluate current folate status in the healthy Kazakh population, we screened serum folate and plasma HCY in 50 healthy Kazakh adults aged 20-65 years (KZH) and 60 healthy Japanese control subjects aged 27-65 years (JPN). RESULTS: Serum levels of folate were significantly lower in KZH than in JPN (3.1 vs 10.0 ng/ml, P<0.01). Fifty of 61 (82.0%) subjects in KZH but none in JPN showed low concentrations of folate (<3.6 ng/ml). Plasma levels of HCY were significantly higher in KZH than in JPN (13.2 vs 7.8 nmol/ml, P<0.01). CONCLUSIONS: Our findings strongly suggest that owing to the insufficient intake of vegetables, folate deficiency exists in Kazakhstan. Furthermore, hyperhomocysteinemia was observed in this group, probably owing to the secondary effects of folate deficiency.

Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and lung metastatic cancer.

We synthesized a potent new antitumor podophyllotoxin derivative (4beta-aminoalkyl-4'-O-demethyl-4-desoxypodophyllotoxin; TOP-53) in our search for a drug that has strong activity against lung cancer and lung metastatic cancer. TOP-53 exhibited twice the inhibitory activity of etoposide (VP-16) against topoisomerase II and induced DNA strand breaks but showed no inhibitory activity against tubulin polymerization. The in vitro cytotoxic activity of TOP-53 assessed as IC50 was 0.016-0.37 microg/ml and 0.26-8.9 microg/ml against marine tumor and human non-small cell lung cancer (NSCLC) cell lines, respectively. TOP-53 exerted significant efficacy equivalent to that of VP-16 on s.c.-implanted murine solid tumors (Colon 26, B16-BL6, and Lewis lung carcinoma) at doses 3-5 times lower than that of VP-16. In human tumor xenografts using NSCLC, TOP-53 was active for four of five tumors, whereas VP-16 was active for two of five tumors. Potent inhibitory activity of TOP-53 was also found against a lung tumor (Lewis lung carcinoma) and four lung metastatic tumors (NL-22 and NL-17 colon cancer, UV2237M fibrosarcoma, and K1735M2 melanoma). TOP-53 appeared to be more active against four of them than VP-16. Thus, TOP-53 is not only active against s.c.-implanted lung cancers but also strongly active against lung localized tumor and metastatic tumors in the lungs. The high selectivity of TOP-53 was attributed to its high distribution into the lung and its persistence. TOP-53 is expected to be highly effective against lung cancer including NSCLC and various lung metastatic tumors in the clinical field.

Effects of iodothyronines on chemotactic peptide-receptor binding and superoxide production of human neutrophils.

We studied the action of iodinated thyronines on the superoxide (O2-) production of human neutrophils stimulated with a chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) in vitro. L-Thyroxine and L-triiodothyronine elicited dose dependently a potent inhibitory action on the FMLP-induced O2- production with IC50 values of about 10(-6) M and 7.10(-6) M, respectively, but L-diiodothyronine did not. No difference in the inhibition was observed between the L-form and the D-form of the compounds. Inhibition of the O2- production by L-thyroxine was restored by the washing of the cells. L-Thyroxine did not affect the O2- production stimulated with either the fifth component of the complement (C5a) or phorbol 12-myristate 13-acetate. L-Thyroxine and L-triiodothyronine were found to block [3H]FMLP binding to its own receptor with IC50 values similar to those for the inhibition of the O2- production by changing the affinity for the peptide but not the number of the receptors. These results suggest that thyroxine and triiodothyronine interfere with the binding of FMLP to the receptors, leading to the inhibition of neutrophil functions, such as O2- production, and that the inhibitory effects result from extranuclear actions rather than nuclear receptor-mediated ones.

Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes.

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of N-acetyl-L-glutamate, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well in parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in N-acetyl-L-glutamate concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides.

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.

Visualization of yellowish-orange luminescence from cuprous metallothioneins in liver of Long-Evans Cinnamon rat.

We describe the first use of an emission probe, based on the cuprous thiolate chromophore, for direct microscopical observation of cuprous metallothioneins located in liver of 15-week-old (just before spontaneous hepatitis) Long-Evans Cinnamon rats. The rats show remarkable accumulations of copper and cuprous metallothioneins. In the mildly fixed liver, we visualized the same yellowish-orange luminescence as the specific emission from cuprous metallothioneins, following excitation in 330-385 nm region. In liver from Long-Evans Agouti rat, a counter part of Long-Evans Cinnamon rat, no similar luminescence was found. So, it was thought that cuprous metallothioneins accumulated in the Long-Evans Cinnamon rat liver might emit the yellowish-orange light. To verify this presumption, we tentatively defined three histochemical criteria, quenching tests by oxidation, protonation and mercury treatment, based on the coordination chemical characteristics of metallothioneins. The emission completely satisfied these criteria. Furthermore, the reliability of these criteria was supported by immunocytochemical and biochemical results. Consequently, all results sufficiently indicate that the yellowish-orange luminescence in the Long-Evans Cinnamon rat liver is the emission from cuprous metallothioneins.