Association of low back pain and sleep quality with presenteeism.

BACKGROUND: Low back pain (LBP) and poor subjective sleep quality (SSQ) are major risk factors for presenteeism. However, no studies have investigated whether combined LBP and poor SSQ are associated with presenteeism. AIMS: We aimed to examine whether a combination of LBP and poor SSQ is associated with presenteeism. METHODS: This cross-sectional study included 936 workers (median age, 38 years; men, 89%), with evaluated presenteeism using the work limitations questionnaire. We divided them into 'no presenteeism' and 'presenteeism' categories. The presence of LBP was defined as a numerical rating scale (NRS) score of ≥1 in current pain intensity. SSQ was assessed using a single question regarding whether the participants typically got enough sleep. We categorized the participants into four groups: (i) LBP + poor SSQ, (ii) non-LBP + poor SSQ, (iii) LBP + good SSQ and (iv) non-LBP + good SSQ. Logistic regression analysis was used to investigate the association between presenteeism and the presence of LBP and poor SSQ, adjusting for age, sex, work type, education, marital status, smoking status, body mass index and weekly working hours. RESULTS: The data from 533 participants were used for analysis (median age, 38 years; men, 90%, response rate, 66%). Combined LBP and poor SSQ were significantly associated with presenteeism (non-LBP + poor SSQ: adjusted odds ratio = 0.56, 95% CI 0.32-0.96; LBP + good SSQ: 0.33, 0.20-0.56; non-LBP + good SSQ: 0.29, 0.18-0.48). CONCLUSIONS: Evaluating both LBP and SSQ may be beneficial in considering presenteeism.

Expression of the Short Form of RON/STK in Feline Mammary Carcinoma.

RON is a tyrosine kinase receptor activated by the macrophage-stimulating protein (MSP) ligand that is overexpressed in human breast cancer. In humans, RON protein can be present in different isoforms, and the most studied isoform is represented by the short form of RON ( sf-RON), which is generated by an alternative promoter located in intron 10 of the RON complementary DNA (cDNA). It plays an important role in breast cancer progression. Considering the many similarities between feline mammary carcinoma (FMC) and human breast cancer, the aim of this study was to investigate the expression of both RON and MSP in FMCs and to identify the presence of the sf-RON transcript. Tissue samples of spontaneous mammary tumors were collected from 60 queens (10 benign lesions, 50 carcinomas). All of the samples were tested for RON and MSP expression by immunohistochemistry; moreover, RNA was extracted from paraffin-embedded tissue samples, and the cDNA was tested by reverse transcription-polymerase chain reaction (RT-PCR) to identify the presence of sf-RON. Immunohistochemistry detected the expression of RON and MSP in 34 of 50 (68%) and 29 of 50 (58%) FMCs, respectively. RT-PCR revealed the presence of the short-form in 18 of 47 (38%) FMCs. This form originates, as in humans, from an alternative promoter (P2), and it codes for the proper feline short form ( sf-RON). sf-RON expression was associated with poorly differentiated tumors and with a shorter disease-free ( P < .05; hazard ratio [HR], 2.2) period and a shorter survival ( P < .05; HR, 2.2). These results support FMC as a suitable model in comparative oncology and identify sf-RON expression as potential predictor of outcomes for this disease.

Pharmacokinetic/pharmacodynamic analysis of adjunctive perampanel in subjects with partial-onset seizures.

OBJECTIVES: Explore perampanel pharmacokinetics (PK) in all subjects (aged ≥12 years) vs adolescents (aged ≥12 to ≤17 years) with partial-onset seizures (POS) and identify factors explaining between-subject variability in efficacy using a population PK/pharmacodynamic (PD) analysis. MATERIALS & METHODS: Population PK analysis was performed using nonlinear mixed-effect modeling with data from phase II/III randomized, double-blind, placebo-controlled studies of adjunctive perampanel in POS. Perampanel exposure was predicted for all subjects and adolescents. Population PK/PD analyses were performed using data from phase III studies to explore the relationship between perampanel exposure and 28-day average seizure frequency and responder probability. RESULTS: Pooled perampanel PK data from 1318 subjects were described by a one-compartment disposition model. In the absence of antiepileptic drugs (AEDs) affecting perampanel PK, estimated perampanel apparent clearance (CL/F) was 0.668 L/h (all subjects) and 0.682 L/h (adolescent subjects). Co-administration of carbamazepine and oxcarbazepine/phenytoin reduced perampanel exposure. Gender, Asian race (excluding Japanese or Chinese), and increasing alanine aminotransferase lowered perampanel CL/F, but differences were small and not considered clinically relevant. Adolescent outcomes were similar to the total population. Based on PK/PD data from 1748 subjects, percent reduction in 28-day average seizure frequency from baseline and responder probability increased with increasing perampanel exposure; concomitant CYP3A-inducing AEDs lowered perampanel exposure but did not impact the slope for responder probability. CONCLUSIONS: These results are consistent with previous analyses but expand on these through inclusion of a larger number of patients from different ethnic groups, and demonstrate that outcomes were similar between adults and adolescents.

Adjunctive perampanel in partial-onset seizures: Asia-Pacific, randomized phase III study.

OBJECTIVES: To evaluate the efficacy, safety, and tolerability of perampanel, a selective, non-competitive, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, as an adjunctive treatment for patients with refractory partial-onset seizures (POS) from Asia-Pacific. MATERIALS & METHODS: This multicenter, randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov identifier: NCT01618695) involved patients aged ≥12 years with refractory POS (receiving 1-3 antiepileptic drugs). Patients were randomized (1:1:1:1) to receive once-daily placebo or perampanel 4, 8, or 12 mg over a 6-week titration and 13-week maintenance double-blind period. Enzyme-inducing antiepileptic drugs were equally stratified between groups. The primary efficacy endpoint was percent change in POS frequency per 28 days (double-blind phase vs baseline). Other efficacy endpoints included ≥50% responder rate and seizure freedom. Treatment-emergent adverse events (TEAEs) were also monitored. RESULTS: Of 710 randomized patients, seizure frequency data were available for 704 patients. Median percent changes in POS frequency per 28 days indicated dose-proportional reductions in seizure frequency: -10.8% with placebo and -17.3% (P = .2330), -29.0% (P = .0003), and -38.0% (P < .0001) with perampanel 4, 8, and 12 mg, respectively. In total, 108 (15.3%) patients discontinued treatment; 44 (6.2%) due to TEAEs. TEAEs occurring in ≥5% of patients, and reported at least twice as frequently with perampanel vs placebo, included dizziness and irritability. CONCLUSIONS: Adjunctive perampanel (8 and 12 mg/d) significantly improved seizure control in patients with refractory POS. Safety and tolerability were acceptable at daily doses of perampanel 4-12 mg.

Repair of dentin defects from DSPP knockout mice by PILP mineralization.

Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp-/-) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the "polymer-induced liquid-precursor" (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp-/- and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp-/- dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair.

Gene silencing of STAT6 with siRNA ameliorates contact hypersensitivity and allergic rhinitis.

BACKGROUND: Silencing of genes using small interfering RNA (siRNA) is a recently developed strategy to regulate the synthesis of target molecules. Signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity. METHODS: To elucidate the therapeutic potential of using siRNA to inhibit STAT6 in allergic reactions, we determined the nucleotide sequences of siRNA specific for STAT6. RESULTS: The selected sequences of STAT6 siRNA specifically inhibited the generation of STAT6 synthesis in dermal fibroblasts and eotaxin (CCL11) production in response to IL-4/TNF-α in vitro. Local administration of STAT6 siRNA in vivo alleviated contact hypersensitivity responses to chemical haptens. This was accompanied by reduced local production of IL-4, IL-13, eotaxin (CCL11), TARC (CCL17) and MDC (CCL22). Similarly, consecutive intranasal instillation of STAT6 siRNA markedly inhibited inflammatory cellular infiltration of mucosal tissues in allergic rhinitis responses in association with reduced IL-4 and IL-5 production from regional lymph node cells. Immediate responses, such as sneezing and nasal rubbing behaviors, were also improved by STAT6 siRNA. CONCLUSIONS: Local administration of STAT6 siRNA is thus a promising therapeutic strategy for both Th2-mediated cutaneous diseases and allergic rhinitis.

Working hours, occupational stress and depression among physicians.

BACKGROUND: Physicians report high prevalence of depression, work long hours and are exposed to many occupational stresses (OSs). AIMS: To investigate the cross-sectional association between working hours, OS and depression among physicians. METHODS: A self-administered questionnaire was mailed to 1902 alumni of a medical school. The questionnaire evaluated working hours in the previous week, OS assessed by the effort-reward imbalance model, social support and depression evaluated by the Center for Epidemiologic Studies Depression scale. The associations between these occupational factors and depression were analyzed using multiple logistic regression. RESULTS: The questionnaire was returned by 795 alumni (response rate, 42%), and 706 respondents (534 men and 172 women) were suitable for analysis. The odds ratio (OR) of depression in the long working hours group (>70 h/week) was 1.8 (95% CI: 1.1-2.8) compared with the short working hours group (<54 h/week), adjusted for basic attributes. The adjusted ORs of depression in the upper effort-reward ratio (ERR) tertile versus the lower ERR tertile were 0.6 (0.2-1.8) in the short working hours group, 8.5 (3.0-24.0) in the middle working hours group and 9.9 (3.8-25.7) in the long working hours group. The adjusted ORs of depression stratified according to working hours and ERR tended to be higher in the groups with a higher ERR, but no association between working hours and depression was found. CONCLUSIONS: This study indicates that the management of OS is needed as a countermeasure against depression among physicians.

Increasing the number of steps walked each day improves physical fitness in Japanese community-dwelling adults.

We aimed to investigate the effects of increasing the number of steps each day on physical fitness, and the change in physical fitness according to the angiotensin-converting enzyme (ACE) genotype. A total of 174 participants were randomly assigned to two groups. Subjects in group A were instructed for 24-week trial to increase the number of steps walked each day, while subjects in group B were instructed to engage in brisk walking, at a target heart rate, for 20 min or more a day on two or more days a week. The values of the 3-min shuttle stamina walk test (SSWT) and the 30-s chair-stand test (CS-30) significantly increased, but no differences in increase were found between the groups. A significant relationship was found between the percentage increase in SSWT values and the increase in the number of steps walked by 1 500 steps or more per day over their baseline values. Our results suggest that increasing the number of steps walked daily improves physical fitness. No significant relationships were observed between the change in physical fitness and ACE genotypes.

Evaluation of epithelial and mesenchymal cell markers in canine urinary bladder transitional cell carcinoma.

Canine transitional cell carcinoma (cTCC) is the most common malignant tumour in the urinary bladder: it is highly invasive and exhibits metastatic characteristics. Inflammation is also strongly related to cTCC. Epithelial tumours often exhibit a mesenchymal cell phenotype during tumour invasion and metastasis owing to epithelial-mesenchymal transition (EMT), which is often induced in chronic inflammation. The aim of this retrospective study was to investigate the expression of epithelial and mesenchymal cell markers in tumour cells and to evaluate its relationship with prognosis of cTCC. In this study, 29 dogs with cTCC who underwent surgical treatment were enrolled. Clinical parameters were reviewed using medical records. Tissue expression of epithelial and mesenchymal markers was evaluated by immunohistochemical analysis. The association between the expression of mesenchymal cell markers and clinical parameters, including prognosis, was statistically examined. In five normal bladder tissues used as controls, no expression of mesenchymal markers was observed, except for one tissue that expressed fibronectin. Conversely, epithelial tumour cells expressed vimentin and fibronectin in 23/29 and 19/28 cTCC tissues, respectively. Regarding clinical parameters, vimentin score in Miniature Dachshunds was significantly higher than those in other dog breeds (P < 0.001). Multivariate survival analyses revealed that age>12 years was related to shorter progression-free survival (P = 0.02). Higher vimentin score, lower fibronectin score, and advanced clinical T stage were significantly correlated with shorter median survival time (P < 0.05). The results of this study indicate that vimentin expression was associated with cTCC progression. Further studies are needed to examine the incidence and relevance of EMT in cTCC.

Relationship between Breakfast Skipping and Obesity among Elderly: Cross-Sectional Analysis of the HEIJO-KYO Study.

OBJECTIVE: Breakfast skipping is reported to be associated with obesity in children and younger populations; however, few studies report the association among elderly. The purpose of this study was to investigate the relationships between breakfast skipping and obesity prevalence among elderly. DESIGN: Cross-sectional study. SETTING: Community-dwelling elderly in Nara, Japan. PARTICIPANTS: 1052 elderly participants (mean age: 71.6 years). MEASUREMENTS: Obesity and breakfast skipping were defined as body mass index of ≥25 kg/m2 and skipping breakfast one or more times per week, respectively. RESULTS: Two hundred and seventy-two participants (25.9%) were classified as obese and forty-one (3.9%) were as breakfast skippers. Obesity prevalence was significantly higher in breakfast skippers than in breakfast eaters (43.9% vs. 25.1%, P = 0.007). In multivariable logistic regression analysis adjusted for potential confounders (age, sex and alcohol consumption), breakfast skippers showed significantly higher odds ratio (OR) for obesity than breakfast eaters (OR, 2.23; 95% confidence interval, 1.17-4.27; P = 0.015), which continued to be significant after further adjustment for socioeconomic status. In addition, breakfast skippers showed significantly lower daily potassium (P <0.001) and dietary fibre intakes (P = 0.001) and lower subjective physical activity (P = 0.035) than breakfast eaters. CONCLUSIONS: Breakfast skipping was significantly associated with obesity among elderly. Poor diet quality and physical inactivity may be potential intermediators underlying the association between breakfast skipping and obesity.

Anti-tumour efficacy of etoposide alone and in combination with piroxicam against canine osteosarcoma in a xenograft model.

Osteosarcoma (OSA) in dogs is locally invasive and highly malignant. Distant metastasis is the most common cause of death. To date, the survival rate in dogs with OSA remains poor. The cytotoxic effects of etoposide against canine OSA cell lines, either alone or in combination with piroxicam, have been previously demonstrated in vitro. The aim of this study was to evaluate the anti-tumour effect of etoposide alone and in combination with piroxicam on canine OSA using murine models. Etoposide single agent treatment significantly delayed tumour progression with a marked reduction in Ki-67 immunoreactivity in tumour tissue. Concomitant treatment with piroxicam did not enhance the anti-tumour efficacy of etoposide. Etoposide single agent treatment and combination treatment with piroxicam down-regulated survivin expression, but was not followed by increased apoptotic activity. These findings indicate that etoposide might be a promising novel therapeutic for canine OSA. Further investigations into its potential for clinical application in veterinary oncology are warranted.

Recovery after PILP remineralization of dentin lesions created with two cariogenic acids.

OBJECTIVES: Acetate and lactate are important cariogenic acids produced by oral bacteria. They produced different residual dentin structures in artificial lesions of similar depth. We evaluated if such lesions responded in the same way to a polymer-induced-liquid-precursor (PILP) remineralization. DESIGN: Dentin blocks obtained from human third molars, divided into 6 groups (n=3). Blocks were demineralized with acetate (66h) or lactate (168h) buffer at pH 5.0 to create 140µm target lesion depths. A-DEM and L-DEM groups received no remineralization. Other groups were remineralized for 14days. 100µg/mL polyaspartate was added into the remineralizing buffer for A-PIL and L-PIL, whereas A-CAP and L-CAP were treated with the same solution but without polyaspartate. Cross-sectioned blocks were examined for shrinkage and AFM-topography. Line profiles of reduced elastic modulus (Er) were obtained by AFM-based nanoindentation across the lesion. Ultrastructures were examined with TEM. RESULTS: A-PIL and L-PIL recovered in shrinkage to the original height of the dentin and it appeared normal with tubules, with increases in Er at both outer flat and inner sloped zones. At the sloped zone, acetate lesions lost more Er but recovery rate after PILP was not statistically different from lactate lesions. A-CAP and L-CAP showed surface precipitates, significantly less recovery in shrinkage or Er as compared to PILP groups. TEM-ultrastructure of PILP groups showed similar structural and mineral components in the sloped zone for lesions produced by either acid. CONCLUSIONS: The PILP process provided significant recovery of both structure and mechanical properties for artificial lesions produced with acetate or lactate.

Molecular investigation of the direct anti-tumour effects of nonsteroidal anti-inflammatory drugs in a panel of canine cancer cell lines.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been suggested as effective adjunctive anti-tumour agents in human and veterinary medicine. However, the molecular mechanisms associated with their anti-tumour effects and correlations with the expression of cyclooxygenase (COX) and related molecules in tumours remain controversial. The objective of this study was to compare the expression profiles of COX and related molecules with NSAID sensitivity and to explore the molecular mechanisms of anti-tumour effects. The expression profiles of COXs, prostaglandins (PGs), PGD2 synthases, and PGE2 synthases were obtained, and their correlations with in vitro sensitivity to the NSAIDs piroxicam, carprofen, and robenacoxib were examined, using 26 canine cancer cell lines. Subsequently, microarray analysis was performed using one melanoma cell line to gain insight into mechanisms by which NSAIDs could exert cytotoxic effects. No strong correlation was observed between the cellular expression of COX and related molecules and sensitivity to NSAID treatment. Additionally, NSAIDs inhibited cell growth only at considerably higher concentrations than those required for functional COX inhibition. Microarray data demonstrated that five genes (SLC16A6, PER2, SLC9A8, HTR2B, and BRAF) were significantly upregulated and that four genes (LOC488305, H2AFJ, LOC476445, and ANKRD43) were significantly downregulated by NSAID exposure to the melanoma cell line. These results suggest that the direct in vitro anti-tumour effects of NSAIDs might be mediated by COX/PG-independent pathways. Novel candidate genes that could potentially be involved in the anti-tumour effects of NSAIDs were identified. Further validation and elucidation of their associated mechanisms will contribute to patient selection in clinical settings and the development of effective combination therapies.

Effects of etoposide alone and in combination with piroxicam on canine osteosarcoma cell lines.

Osteosarcoma (OSA) is the most common primary bone tumour in dogs. The poor survival rate in dogs with OSA highlights the need for new therapeutic approaches. This study evaluated the cytotoxic effects of etoposide, alone and in combination with piroxicam, on canine OSA cell cultures. Etoposide alone significantly suppressed cell growth and viability, whereas etoposide in combination with piroxicam exhibited concentration dependent cytotoxicity. The anti-proliferative effect was a result of inactivity of the Cdc2-cyclin B1 complex, which correlated with an increase in the G2/M fraction. This subsequently activated the apoptosis cascade, as indicated by elevated apoptosis levels and up-regulation of poly (ADP-ribose) polymerase proteolytic cleavage. Down-regulation of survivin expression induced by the combination treatment may have contributed to the enhanced cytotoxicity. The results of this study suggest that further investigation of etoposide and piroxicam as a therapeutic combination for canine OSA is warranted.

Distinct decalcification process of dentin by different cariogenic organic acids: Kinetics, ultrastructure and mechanical properties.

OBJECTIVES: We studied artificial dentin lesions in human teeth generated by lactate and acetate buffers (pH 5.0), the two most abundant acids in caries. The objective of this study was to determine differences in mechanical properties, mineral density profiles and ultrastructural variations of two different artificial lesions with the same approximate depth. METHODS: 0.05M (pH 5.0) acetate or lactate buffer was used to create 1) 180µm-deep lesions in non-carious human dentin blocks (acetate 130h; lactate 14days); (2) demineralized, ∼180µm-thick non-carious dentin discs (3 weeks). We performed nanoindentation to determine mechanical properties across the hydrated lesions, and micro X-ray computed tomography (MicroXCT) to determine mineral profiles. Ultrastructure in lesions was analyzed by TEM/selected area electron diffraction (SAED). Demineralized dentin discs were analyzed by small angle X-ray scattering (SAXS). RESULTS: Diffusion-dominated demineralization was shown based on the linearity between lesion depths versus the square root of exposure time in either solution, with faster kinetics in acetate buffer. Nanoindentation revealed lactate induced a significantly sharper transition in reduced elastic modulus across the lesions. MicroXCT showed lactate demineralized lesions had swelling and more disorganized matrix structure, whereas acetate lesions had abrupt X-ray absorption near the margin. At the ultrastructural level, TEM showed lactate was more effective in removing minerals from the collagenous matrix, which was confirmed by SAXS analysis. CONCLUSIONS: These findings indicated the different acids yielded lesions with different characteristics that could influence lesion formation resulting in their distinct predominance in different caries activities, and these differences may impact strategies for dentin caries remineralization.

Sequence analysis and expression of a novel mouse homolog of Escherichia coli recA gene.

Escherichia coli recA and its yeast homologs RAD51 and DMC1 play crucial roles in mitotic and/or meiotic recombination and in repair of double-strand DNA breaks. We have identified a murine novel recA-like gene (MmTRAD). The predicted 329 amino acid protein showed significant homology to mouse Rec2, Rad51, Dmc1 (or Lim15) and E. coli RecA. Northern blot analysis revealed that MmTRAD was ubiquitously transcribed in various tissues.

Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond.

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.

Purification and characterization of an alkaline amylopullulanase with both alpha-1,4 and alpha-1,6 hydrolytic activity from alkalophilic Bacillus sp. KSM-1378.

The novel alkaline amylopullulanase produced by alkalophilic Bacillus sp. KSM-1378 was purified to an electrophoretically homogeneous state from culture medium. The purified enzyme was a glycoprotein with an apparent molecular mass of about 210 kDa and an isoelectric point of pH 4.8. The N-terminal amino acid sequence was Glu-Thr-Gly-Asp-Lys-Arg-Ile-Glu-Phe-Ser-Tyr-Glu-Arg-Pro and showed no homology to the N-terminal regions of other amylopullulanases reported to date. The enzyme was able to attack specifically the alpha-1,6 linkages in pullulan to generate maltotriose as the major end product, as well as the alpha-1,4 linkages in amylose, amylopectin and glycogen to generate various oligosaccharides. The pH and temperature optima for the pullulanase and alpha-amylase activities were pH 9.5 and 50 degrees C and pH 8.5 and 50 degrees C respectively. Both activities were strongly inhibited by well characterized inhibitors, such as diethyl pyrocarbonate and N-bromosuccinimide. The pullulanase activity was specifically inactivated by Hg2+ ions, alpha-cyclodextrin and beta-cyclodextrin while the amylase activity was strongly inhibited by EDTA and EGTA, although inhibition could be reversed by Ca2+ ions. It is suggested that the single alkaline amylopullulanase protein has two different active sites, one for the cleavage of alpha-1,4-linked substrates and one for the cleavage of alpha-1,6-linked substrates.

Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells.

To understand the roles of bcl-2 for the survival of leukemic cells, we constructed human leukemic HL60 transformant lines in which full length bcl-2 antisense message was conditionally expressed by a tetracycline-regulatable expression system. Cell growth was completely inhibited after antisense message induction and massive cell death was induced. Electron microscopic examinations show that cells died by autophagy, but not by apoptosis. The morphology and the function of mitochondria remained intact: neither the reduction in mitochondrial membrane potential nor the nuclear translocation of AIF, a mitochondrial protein that translocates to nuclei in cases of apoptosis, was observed. Caspase inhibitors did not rescue bcl-2-antisense-mediated autophagy. Thus, bcl-2 is essential for leukemic cell survival and its down-regulation results in autophagy. Cell Death and Differentiation (2000) 7, 1263 - 1269.

Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody.

The position of the N terminus of myosin light chain 1 (LC1) and myosin light chain 2 (LC2) of rabbit skeletal muscle was mapped on the myosin head with a monoclonal antibody (SI304), which recognized the amino acid sequence N-trimethylalanyl-prolyl-lysyl-lysyl at the N terminus of LC1 and LC2. The complex of the antibody and myosin was observed by electron microscopy. By selective cleavage of the N terminus of LC1 or LC2 with papain or chymotrypsin, the position of the N terminus of LC1 and LC2 was determined separately. The N terminus of LC2 is located at the head-rod junction. The N terminus of LC1 is 11 nm (+/- 3 nm, standard deviation) from the head-rod junction. This position is near the actin-binding site of the myosin head.