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1.
Occup Med (Lond) ; 72(7): 439-445, 2022 10 18.
Article in English | MEDLINE | ID: mdl-35657324

ABSTRACT

BACKGROUND: The education sector has been heavily impacted by COVID-19. While the impact on school-aged children has received much attention, less attention has focused on the experiences of educators. AIMS: To compare various dimensions of the psychosocial work environment and health outcomes between educators engaged in online learning to those engaged in in-person learning in the Canadian province of Ontario. METHODS: Responses from 5438 educators engaged in either online or in-person learning were collected between 23 November and 21 December 2020; three months after the start of the 2020/21 academic year in September 2020. Psychosocial outcomes included quantitative demands, work pace, predictability, role conflicts, and social support from supervisors and co-workers; assessed using an abbreviated version of the Copenhagen Psychosocial Questionnaire. Secondary outcomes included burnout and sleep troubles. Ordinary Least-Squares regression models examined adjusted mean differences in the levels of outcomes for respondents in in-person versus online learning, after adjustment for a variety of covariates. RESULTS: Compared to respondents engaged in in-person learning, respondents engaged in online learning reported less predictability, higher role conflicts and less support from supervisors and co-workers. Statistically significant differences in work pace, burnout and sleep troubles were also observed across learning modes, although these differences did not exceed previously suggested thresholds for minimum important differences. CONCLUSIONS: Important differences in the psychosocial work environment were observed between respondents engaged in in-person learning versus online learning. Addressing these differences is required, given the potential continued importance of online learning within the context of the COVID-19 pandemic and beyond.


Subject(s)
Burnout, Professional , COVID-19 , Child , Humans , COVID-19/epidemiology , Pandemics , Workplace/psychology , Burnout, Professional/epidemiology , Ontario/epidemiology
2.
Gait Posture ; 66: 155-159, 2018 10.
Article in English | MEDLINE | ID: mdl-30195218

ABSTRACT

BACKGROUND: As the proportion of older adults in the population increases, so does the associated prevalence of falls, making falls the leading cause of fatal and nonfatal injuries among adults aged ≥65 years. In response, researchers and clinicians seek to develop a clinical tool that accurately predicts fall risk. These Investigations have included measures of clinical mobility and balance tests, strength, physiologically based tests, postural sway, and mean and variability of gait measures. To date, no study has concurrently explored all these measures to determine which measures, alone or in combination, emerge as the most predictive of fall risk. While there is evidence that dual-task gait conditions are sensitive indicators of fall risk, difference scores between dual-task and single-task gait conditions (DS) have not been explored. RESEARCH QUESTION: This study included outcome measures representing diverse domains (clinical mobility and balance, strength, physiological, postural sway, and mean and variability of difference scores between dual- and single-task gait conditions) to determine the combination of measures that were the most sensitive for retrospectively classifying fallers from non-fallers. METHODS: Forty-two (mean: 75.8 yrs ± 3.3) community-dwelling older adults completed a comprehensive battery of 76 measures and classified into two groups based on self-report of having one or more falls in the previous year. RESULTS: Results suggest that 11 measures captured the salient characteristics of the total cohort (fallers (N = 27) and non-fallers (N = 15) and that five gait measures were sufficient for correctly classifying fallers and non-fallers with 92.3% sensitivity and 66.7% specificity with a total model classification of 82.9%. SIGNIFICANCE: The five variables comprise mean DS of stride timing, stride width, and stride length and DS in variability for stride width and stride velocity demonstrating that difference in performance between dual-task and single-task gait trials was essential for discriminating fallers and superior to other measures.


Subject(s)
Accidental Falls/prevention & control , Gait/physiology , Risk Assessment/methods , Aged , Aged, 80 and over , Aging/physiology , Analysis of Variance , Cohort Studies , Female , Humans , Logistic Models , Male , Postural Balance/physiology , Principal Component Analysis , Retrospective Studies , Sensitivity and Specificity
3.
Genome ; 49(6): 619-30, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16936841

ABSTRACT

Rice (Oryza sativa L.) pathogenesis-related (PR)-3 chitinases, like other PR proteins, are each coded by one of the genes of a multigene family in the plant genome. We assembled the database information about rice PR-3 chitinase sequences. A total of 12 PR-3 chitinase loci (Cht1 to Cht12) were found deployed in the rice genome. Some of the loci were occupied by 2 or more alleles. For all the loci expect Cht4, Cht5, Cht6, and Cht11, the amino acid sequence was polymorphic between japonica and indica varieties of rice, but glutamic acid acting as a catalytic residue was completely conserved in all the loci expect Cht7. All the genes except Cht7, which was not tested in this study, were transcripted in some organs (leaf, sheath, root, and meristem) of rice plants. These results suggest that chitinase proteins encoded by the genes at these loci have important biological effects, at least antifungal activities, on rice plants. We also proposed a new classification of rice PR-3 chitinases based on their domain structures. This classification was consistent with the results of phylogenetic analysis of rice chitinases.


Subject(s)
Chitinases/genetics , Chitinases/metabolism , Oryza/genetics , Oryza/metabolism , Alleles , Amino Acid Sequence , Base Sequence , Chitinases/classification , Chromosome Mapping , Gene Expression , Molecular Sequence Data , Multigene Family , Organ Specificity , Phylogeny , Plant Proteins/classification , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Homology, Amino Acid , Software , Tissue Distribution
4.
Lett Appl Microbiol ; 40(5): 329-34, 2005.
Article in English | MEDLINE | ID: mdl-15836734

ABSTRACT

AIMS: To investigate the mutational effect for the stability of thermolysin (TLN) in conserved regions. METHODS AND RESULTS: Mutational effects for stability at autodegradation sites of TLN in conserved region were studied. The bands of mutant TLN (34 kDa) on SDS-PAGE were decreased. However, those of mutant TLN cultivated with CaCl2 recovered to the same level as WT TLN. Dialysis study shows that these mutant TLN require more calcium ions than WT TLN. CONCLUSIONS: From these results, calcium affinity of mutant TLN in the conserved regions seem to become weak, subsequently mutant TLN were easily autodegraded in the case of low concentration of CaCl2. SIGNIFICANCE AND IMPACT OF THE STUDY: The autodegradation sites located in conserved regions of bacilli neutral proteases are important for the tertiary structure formation concerning the stability of the protein.


Subject(s)
Bacillus/genetics , Thermolysin/genetics , Amino Acid Sequence , Bacillus/growth & development , Bacillus/metabolism , Calcium Chloride , Cloning, Molecular , Culture Media , Industrial Microbiology , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Sequence Alignment , Thermolysin/chemistry , Thermolysin/metabolism
5.
Lett Appl Microbiol ; 39(1): 89-92, 2004.
Article in English | MEDLINE | ID: mdl-15189293

ABSTRACT

AIMS: To prove that Bacillus thuringiensis serovar shandongiensis strain 89-T-34-22 produces several novel cytotoxic proteins against human leukaemic T cells. METHODS AND RESULTS: Parasporal inclusion protein was solubilized and processed by proteinase K and was separated by anion-exchange chromatography. Cytopathic effects of each fraction against MOLT-4 and Jurkat cells were monitored. CONCLUSIONS: Existence of at least two novel cytotoxic proteins was suggested and N-terminal sequences of the newly identified proteins were determined to be QSTTDVIREY and X (Y or I) (P or I) NLANELA (X indicates uncertain amino acids). Molecular masses of the two proteins were approx. 27-28 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we demonstrated that the strain 89-T-34-22 produces at least two novel cytotoxic proteins with similar molecular masses against human cancer cells. This is the first strain of B. thuringiensis which produces multiple cytotoxic proteins against human cancer cells.


Subject(s)
Antineoplastic Agents/toxicity , Bacillus thuringiensis/metabolism , Bacterial Proteins/toxicity , Cytotoxins/toxicity , Amino Acid Sequence , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cytotoxins/chemistry , Cytotoxins/metabolism , HeLa Cells/drug effects , Humans , Jurkat Cells , Leukemia, T-Cell , Tumor Cells, Cultured/drug effects
6.
Minerva Endocrinol ; 28(2): 87-102, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12717340

ABSTRACT

Patients and animals with poorly controlled or uncontrolled diabetes present with diurnal hypersecretion of glucocorticoids and altered regulation of the hypothalamo-pituitary-adrenocortical (HPA) axis. Although some of these changes are reversed with insulin replacement therapy, neuroendocrine function is not always restored to normal, even with rigorous glycemic control. In addition, stress responsiveness is also impaired in diabetes and this has important implications in the way patients with diabetes cope with many stress challenges, including the metabolic challenge of insulin-induced hypoglycemia. HPA dysregulation in diabetes appears to involve complex interactions between impaired glucocorticoid negative feedback sensitivity and factors such as hypoinsulinemia, hyperglycemia and/or hypoleptinemia, that may increase central drive of the axis. This review examines some of the evidence indicating hyperactivation of the HPA axis in patients with diabetes. Using the streptozotocin-diabetic rat as a model of type-1 diabetes, we will focus on elucidating some of the mechanisms underlying HPA dysregulation in diabetes. Hyperactivation of the HPA axis in diabetes is associated with increased expression of hypothalamic corticotrophin-releasing hormone (CRH) mRNA and hippocampal mineralocorticoid receptor (MR) mRNA. Although insulin replacement restores ACTH and corticosterone levels to normal, likely through glucocorticoid-mediated suppression of ACTH secretion, CRH and MR mRNA expression remain elevated. A better understanding of these mechanisms may be important in developing new treatment modalities for patients with diabetes mellitus.


Subject(s)
Diabetes Mellitus/physiopathology , Hypothalamo-Hypophyseal System/physiopathology , Pituitary-Adrenal System/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Humans
7.
Diabetes Nutr Metab ; 15(5): 348-55; discussion 355-6, 362, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12625483

ABSTRACT

In summary, our data suggest that in uncontrolled diabetes, increased HPA activity is caused by increased central drive at or above the level of the PVN. Insulin treatment only restores HPA activity at and below the pituitary level, presumably by GC-mediated suppression of ACTH secretion. We hypothesize that the defective HPA response to hypoglycaemia is at least in part due to a lack of a decrease in MR mRNA in response to hypoglycaemia, and diminished sensitivity of the pituitary and adrenal gland to stimulation. Interestingly, insulin treatment restores the HPA response, but not the defective epinephrine response. Therefore, defective epinephrine responses are not linked to defective HPA responses. Similarly, antecedent hypoglycaemia specifically impairs epinephrine responses, but not HPA responses to hypoglycaemia. These studies have revealed some of the mechanisms of impaired HPA function in diabetes and its impaired responsiveness to hypoglycaemia. Further investigations are essential for understanding poor counterregulation in insulin-treated diabetes and may lead to new strategies for preventing hypoglycaemia.


Subject(s)
Diabetes Mellitus/physiopathology , Hypoglycemia/physiopathology , Hypothalamo-Hypophyseal System/physiopathology , Pituitary-Adrenal System/physiopathology , Adrenocorticotropic Hormone/physiology , Animals , Corticotropin-Releasing Hormone/physiology , Diabetes Mellitus/metabolism , Epinephrine/blood , Humans , Hypoglycemia/metabolism
8.
J Biochem ; 130(6): 783-8, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11726278

ABSTRACT

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degrees C. Substitution of the active site zinc with other transition metals alters the activity of thermolysin [Holmquist, B. and Vallee, B.L. (1974) J. Biol. Chem. 249, 4601-4607]. Cobalt is the most effective among the transition metals and doubles the activity toward N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide. In this study, the effect of NaCl on the activity of cobalt-substituted thermolysin was examined. Cobalt-substituted thermolysin, with 2.8-fold increased activity compared with the native enzyme, is further activated by the addition of NaCl in an exponential fashion, and the activity is enhanced 13-15 times at 4 M NaCl. The effects of cobalt-substitution and the addition of salt are independent of each other. The activity of cobalt-substituted thermolysin, expressed as k(cat)/K(m), is pH-dependent and controlled by at least two ionizing residues with pK(a) values of 6.0 and 7.8, the acidic pK(a) being slightly higher compared to 5.6 of the native enzyme. These pK(a) values remain constant in the presence of 4 M NaCl, indicating that the electrostatic environment of cobalt-substituted thermolysin is more stable than that of the native enzyme, the acidic pK(a) of which shifts remarkably from 5.6 to 6.7 at 4 M NaCl. Zincov, a competitive inhibitor, binds more tightly to the cobalt-substituted than to native thermolysin at pH 4.9-9.0, probably because of its preference for cobalt in the fivefold coordination. The cobalt substitution has been shown to be a favorable tool with which to explore the active-site microenvironment of thermolysin.


Subject(s)
Cobalt/metabolism , Dipeptides/metabolism , Thermolysin/metabolism , Zinc/metabolism , Acrylates/metabolism , Binding Sites/physiology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Hydrolysis/drug effects , Hydroxamic Acids/pharmacology , Kinetics , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Protease Inhibitors/pharmacology , Sodium Chloride/pharmacology , Substrate Specificity , Thermolysin/antagonists & inhibitors
9.
Eur J Biochem ; 268(24): 6607-15, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11737215

ABSTRACT

We have determined eight types of missense mutants of CYP27B1 from Japanese vitamin D-dependent rickets type I (VDDR-I) patients [Kitanaka, S., Takeyama, K., Murayama, A., Sato, T., Okumura, K., Nogami, M., Hasegawa, Y., Niimi, H., Yanagisawa, J., Tanaka, T. & Kato, S. (1998) New England J. Med., 338, 653-661 and Kitanaka, S., Murayama, A., Sakaki, T., Inouye, K., Seino, Y., Fukumoto, S., Shima, M., Yukizane, S., Takayanagi, M., Niimi, H., Takeyama, K. & Kato, S. (1999) J. Clin. Endocrine Metab., 84, 4111-4117]. None of the CYP27B1 mutants showed 1alpha-hydroxylase activity towards 25-hydroxyvitamin D3. Thus, it was assumed that the mutated amino-acid residues play important roles in the 1alpha-hydroxylase activity, such as substrate binding, activation of molecular oxygen, interaction with adrenodoxin, and folding of the cytochrome P450 structure. To examine our hypothesis, we generated various mutants of CYP27B1 and studied their enzymatic properties. In addition, the corresponding mutations were introduced to CYP27A1, which belongs to the same family as CYP27B1. As CYP27A1 showed much higher expression level than CYP27B1 in Escherichia coli, further analysis including heme-binding and substrate-binding was performed with CYP27A1 in place of CYP27B1. Western blot analysis, spectral analysis including reduced CO-difference spectra and substrate-induced difference spectra, and enzymatic analysis of the mutant CYP27A1 gave information on the structure-function relationships of both CYP27A1 and CYP27B1. Although the sequence alignment suggested that Arg107, Gly125, and Pro497 of CYP27B1 might be involved in substrate binding, the experimental data strongly suggested that mutations of these amino-acid residues destroyed the tertiary structure of the substrate-heme pocket. It was also suggested that Arg389 and Arg453 of CYP27B1 were involved in heme-propionate binding, and Asp164 stabilized the four-helix bundle consisting of D, E, I and J helices, possibly by forming a salt bridge. Thr321 was found to be responsible for the activation of molecular oxygen.


Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase/chemistry , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Rickets/enzymology , Steroid Hydroxylases/chemistry , Steroid Hydroxylases/metabolism , Vitamin D Deficiency/enzymology , Xanthomatosis, Cerebrotendinous/enzymology , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Amino Acid Sequence , Cholestanetriol 26-Monooxygenase , Cytochrome P-450 Enzyme System/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Rickets/etiology , Sequence Homology, Amino Acid , Steroid Hydroxylases/genetics , Structure-Activity Relationship , Vitamin D Deficiency/complications
10.
FEBS Lett ; 507(3): 295-8, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11696358

ABSTRACT

The mechanism of Ras-induced Raf-1 activation is not fully understood. Previously, we identified a 400-kDa protein complex as a Ras-dependent Raf-1 activator. In this study, we identified B-Raf as a component of this complex. B-Raf was concentrated during the purification of the activator. Immunodepletion of B-Raf abolished the effect of the activator on Raf-1. Furthermore, B-Raf and Ras-activated Raf-1 co-operatively, when co-transfected into human embryonic kidney 293 cells. On the other hand, Ras-dependent extracellular signal-regulated kinase/mitogen-activated protein kinase kinase stimulator (a complex of B-Raf and 14-3-3) failed to activate Raf-1 in our cell-free system. These results suggest that B-Raf is an essential component of the Ras-dependent Raf-1 activator.


Subject(s)
Proto-Oncogene Proteins c-raf/metabolism , ras Proteins/metabolism , Cells, Cultured , Humans , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/immunology , Transfection
11.
Biotechnol Annu Rev ; 7: 179-94, 2001.
Article in English | MEDLINE | ID: mdl-11686044

ABSTRACT

The key enzymes of vitamin D3 metabolism, renal 25-hydroxyvitamin D3 1 alpha-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24) were expressed in Escherichia coli, and their enzymatic properties were revealed. As expected, mouse CYP27B1 and human CYP27B1 showed the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with the Michaelis constant, Km, value of 2.7 microM. Unexpectedly, both mouse CYP27B1 and human CYP27B1 showed greater Vmax/Km values toward 24,25-dihydroxyvitamin D3 than 25-hydroxyvitamin D3, suggesting that 24, 25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for both CYP27B1. Enzymatic studies on substrate specificity of CYP27B1 revealed that 25-hydroxyl group of vitamin D3 was essential for the 1 alpha-hydroxylase activity, and 24-hydroxyl group enhanced the activity, but, 23-hydroxyl group greatly reduced the activity. On rat CYP24, it was demonstrated that CYP24 catalyzed four-step monooxygenation towards 25-hydroxyvitamin D3. Furthermore, in vivo and in vitro metabolic studies on 1 alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyzed six-step monooxygenation to convert 1 alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1 alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is highly responsible for the metabolism of both 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3. In addition, we have succeeded in the construction of mitochondrial P450 electron transport chain consisting of ADR, ADX and each of CYP27B1 and CYP24 in E. coli cells. The coexpression system with CYP27B1 might be useful as a bioreactor to produce 1 alpha,25-dihydroxyvitamin D3. In contrast, the coexpression system with CYP24 would be applied to metabolic studies of vitamin D analogs used as drugs.


Subject(s)
25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Cytochrome P-450 Enzyme System/metabolism , Steroid Hydroxylases/metabolism , Vitamin D/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/genetics , Adrenodoxin/genetics , Adrenodoxin/metabolism , Animals , Biotechnology , Cloning, Molecular , Cytochrome P-450 Enzyme System/genetics , Ferredoxin-NADP Reductase/genetics , Ferredoxin-NADP Reductase/metabolism , Humans , In Vitro Techniques , Kinetics , Mice , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Steroid Hydroxylases/genetics , Substrate Specificity , Vitamin D3 24-Hydroxylase
12.
Endocrinology ; 142(11): 4872-9, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11606455

ABSTRACT

Increased hypothalamo-pituitary-adrenocortical (HPA) activity in diabetes is likely important in the development of some pathologies associated with the disorder. We hypothesized that central regulation of HPA activity differs among normal, streptozotocin (STZ)-diabetic, and insulin-treated diabetic rats. Blood glucose, ACTH, and corticosterone were elevated, 8 d after inducing diabetes. Insulin treatment normalized these parameters. Plasma norepinephrine was similar in all groups, but epinephrine was lower in STZ-diabetic and higher in insulin-treated rats vs. normals. Increased ACTH with diabetes corresponded with increased hypothalamic CRH mRNA, but no change in pituitary POMC mRNA. With insulin-treatment, CRH mRNA remained elevated, and POMC mRNA was unaltered. Hippocampal MR mRNA expression was dramatically increased with diabetes and, moreover, was not normalized by insulin. No differences in GR mRNA were detected between normal and STZ-diabetic rats. However, insulin treatment increased GR mRNA levels in the paraventricular nucleus and pituitary. We postulate that, in STZ-diabetes: 1) increased HPA activity is caused by increased central drive at and/or above the level of the paraventricular nucleus and is associated with decreased epinephrine; and 2) normalized pituitary-adrenal activity with insulin may be caused by the compensatory increase in GR mRNA allowing glucocorticoid-mediated suppression of ACTH secretion despite the residual increase in central HPA activity. Thus, insulin apparently restored HPA activity at and below the pituitary but, surprisingly, not above it.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/physiopathology , Hypoglycemic Agents/therapeutic use , Hypothalamo-Hypophyseal System/physiopathology , Insulin/therapeutic use , Pituitary-Adrenal System/physiopathology , Animals , Body Weight , Corticotropin-Releasing Hormone/genetics , Diabetes Mellitus, Experimental/pathology , Hormones/blood , Hypothalamo-Hypophyseal System/drug effects , Male , Pituitary-Adrenal System/drug effects , Pro-Opiomelanocortin/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Steroid/genetics
13.
Am J Physiol Endocrinol Metab ; 281(3): E455-65, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11500300

ABSTRACT

This study aimed to differentiate the effects of repeated antecedent hypoglycemia, antecedent marked hyperinsulinemia, and antecedent increases in corticosterone on counterregulation to subsequent hypoglycemia in normal rats. Specifically, we examined whether exposure to hyperinsulinemia or elevated corticosterone per se could impair subsequent counterregulation. Four groups of male Sprague-Dawley rats were used: 1) normal controls (N) had 4 days of sham antecedent treatment; 2) an antecedent hypoglycemia group (AH) had 7 episodes of hyperinsulinemic hypoglycemia over 4 days; 3) an antecedent hyperinsulinemia group (AE) had 7 episodes of hyperinsulinemic euglycemia; and 4) an antecedent corticosterone group (AC) had 7 episodes of intravenous corticosterone to simulate the hypoglycemic corticosterone levels in AH rats. On day 5, hyperinsulinemic euglycemic-hypoglycemic clamps were performed. Epinephrine responses to hypoglycemia were impaired (P < 0.05 vs. N) after antecedent hypoglycemia and hyperinsulinemia. This correlated with diminished (P < 0.05 vs. N) absolute glucose production responses in AH rats and diminished incremental glucose production responses in AE rats. Paradoxically, norepinephrine responses were increased (P < 0.05 vs. N) after antecedent hypoglycemia. Glucagon and corticosterone responses were unaffected by antecedent hypoglycemia and hyperinsulinemia. In AC rats, incremental but not absolute glucose production responses were decreased (P < 0.05 vs. N). However, neuroendocrine counterregulation was unaltered. We conclude that both antecedent hypoglycemia and hyperinsulinemia impair epinephrine and glucose production responses to subsequent hypoglycemia, suggesting that severe recurrent hyperinsulinemia may contribute to the development of hypoglycemia-associated autonomic failure.


Subject(s)
Corticosterone/pharmacology , Homeostasis , Hyperinsulinism/physiopathology , Hypoglycemia/physiopathology , Animals , Blood Glucose/metabolism , Body Weight , Corticosterone/administration & dosage , Corticosterone/blood , Glucagon/blood , Glucose/biosynthesis , Glucose Clamp Technique , Hypoglycemia/chemically induced , Insulin , Kinetics , Male , Norepinephrine/blood , Rats , Rats, Sprague-Dawley
14.
J Cell Sci ; 114(Pt 11): 2155-65, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11493651

ABSTRACT

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc(-) cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general. Movies available on-line


Subject(s)
Actins/antagonists & inhibitors , Cell Surface Extensions/drug effects , Cytoskeleton/drug effects , Dictyostelium/cytology , Dictyostelium/drug effects , Myosin Type II/metabolism , Quinine/pharmacology , Actins/metabolism , Animals , Biopolymers/antagonists & inhibitors , Biopolymers/metabolism , Cell Movement/drug effects , Cell Size/drug effects , Cell Surface Extensions/metabolism , Cytoskeleton/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Osmolar Concentration
15.
J Biochem Biophys Methods ; 48(1): 23-32, 2001 Mar 28.
Article in English | MEDLINE | ID: mdl-11282399

ABSTRACT

In the preparation of F(ab')(2) fragments of monoclonal antibodies (mAbs) of IgG class, heavy (H) chains are truncated by pepsin and light (L) chains are remained intact. However, F(ab')(2) fragments formed by pepsin-digestion of a mouse mAb PM373, which was of the IgG1 class and raised against human prostate specific antigen (PSA), indicated that the L chains of 31 kDa were cleaved into 23-kDa fragments as well as the cleavage of H chains of 50 kDa into 28-kDa fragments. On the other hand, F(ab')(2) fragments formed by digesting the mAb by cathepsin D showed that the L chains were intact and the H chains were truncated. The immunoreactivities against PSA of the F(ab')(2) fragments containing the intact L chains and those containing the truncated L chains were almost the same as that of the parental mAb, suggesting that the truncation of the L chains does not affect the interaction of the mAb with its specific antigen.


Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/metabolism , Immunoglobulin Light Chains/metabolism , Pepsin A/metabolism , Animals , Antibodies, Monoclonal/chemistry , Cathepsin D/chemistry , Cathepsin D/metabolism , Chromatography, High Pressure Liquid , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Immunoglobulin Light Chains/chemistry , Mice , Pepsin A/chemistry , Prostate-Specific Antigen/immunology
16.
J Biochem Biophys Methods ; 47(3): 177-88, 2001 Feb 26.
Article in English | MEDLINE | ID: mdl-11245889

ABSTRACT

The binding of Cry1Ac, an insecticidal protein of Bacillus thuringiensis, to a brush border membrane (BBM) isolated from midguts of the diamondback moth Plutella xylostella was examined by surface plasmon resonance (SPR)-based biosensor. BBM was mixed with 1,3-ditetradecylglycero-2-phosphocholine (PC14), a neutral charged artificial lipid, and was reconstructed to a monolayer on a hydrophobic chip for the biosensor. The binding of Cry1Ac to the reconstructed monolayer was analyzed by a two-state binding model, and it was shown that Cry1Ac bound to the monolayer in the first step with an affinity constant (K(1)) of 508 nM, followed by the second uni-molecular step with an equilibrium constant (K(2)) of 0.472. The overall affinity constant K(d) was determined to be 240 nM. The binding was markedly inhibited by N-acetyl-D-galactosamine (K(i)=8 mM). The monolayer was shown to retain a high affinity to Cry1Ac, providing an insect-free system for rapid and large-scale screening of B. thuringiensis insecticidal proteins by the SPR-based biosensor technology.


Subject(s)
Bacterial Proteins/metabolism , Bacterial Toxins , Endotoxins/metabolism , Insect Proteins , Surface Plasmon Resonance , Animals , Bacillus thuringiensis/chemistry , Bacillus thuringiensis Toxins , CD13 Antigens/metabolism , Digestive System/metabolism , Hemolysin Proteins , In Vitro Techniques , Membranes, Artificial , Microscopy, Electron , Microvilli/metabolism , Moths/metabolism , Receptors, Cell Surface/metabolism
17.
J Biochem ; 129(3): 429-35, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11226883

ABSTRACT

The effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (K(i)) of thiorphan and R-94138 for matrilysin at pH 7.5, 25 degrees C were determined to be 11.2 and 7.65 microM, respectively. From the temperature dependence of the K(i) values at pH 7.5, the standard enthalpy change (Delta H degrees ') values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2 +/- 0.9) and (1.65 +/- 1.07) kJ x mol(-1), respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the K(i) value suggests that at least two ionizing groups with pK(a) values of 4.5 and 9.1--9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pK(a) values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.


Subject(s)
Acetamides/metabolism , Matrix Metalloproteinase 7/metabolism , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/metabolism , Thiorphan/metabolism , Acetamides/chemistry , Acetamides/pharmacology , Drug Design , Enzyme Stability , Glycopeptides/pharmacology , Humans , Hydrogen-Ion Concentration , Hydroxamic Acids/pharmacology , Kinetics , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding , Static Electricity , Structure-Activity Relationship , Temperature , Thermodynamics , Thiorphan/chemistry , Thiorphan/pharmacology
18.
Biochem Biophys Res Commun ; 280(5): 1346-51, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11162677

ABSTRACT

Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively. These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1. These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM). Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM Triton X-100. On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems. In the fused system, a mixed-type inhibition was observed with K(i) and K(i)' values of 1.2 and 5.4 mM of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system. Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents.


Subject(s)
Cytochrome P-450 CYP1A1/metabolism , Detergents/pharmacology , Animals , Cholic Acids/pharmacology , Cytochrome P-450 CYP1A1/antagonists & inhibitors , Cytochrome P-450 CYP1A1/genetics , Dose-Response Relationship, Drug , Gene Expression Regulation, Enzymologic , Kinetics , Microsomes/drug effects , Microsomes/metabolism , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Octoxynol/pharmacology , Oxazines/metabolism , Rats , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics
19.
J Biochem ; 128(5): 785-91, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11056391

ABSTRACT

Effects of dimethyl sulfoxide (DMSO), temperature, and sodium chloride on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2, 4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. DMSO inhibited the matrilysin activity competitively with the inhibitor constant (K(i)) of 0. 59+/-0.04 M, and the binding between them was endothermic and entropy-driven. The binding of matrilysin with MOCAc-PLGL(Dpa)AR was also found to be entropy-driven. The matrilysin activity was increased in a biphasic exponential fashion with increasing concentration of NaCl, and was 5.3 times higher in the presence of 4 M NaCl than that in its absence. The first and second phases were separated at 0.5 M NaCl, and the activation at x M NaCl compared with the activity in the absence of NaCl was expressed as 2.1(x) at [NaCl] < 0.5 M and 1.4(x) at [NaCl] > 0.5 M. The activation was brought about solely through a decrease in the Michaelis constant (K(m)), and the catalytic constant (k(cat)) was not much altered. This suggests that the decrease in the electrostatic interaction and the increase in the hydrophobic interaction between matrilysin and the substrate might enhance the enzyme activity by reducing the K(m) value.


Subject(s)
Dimethyl Sulfoxide/pharmacology , Matrix Metalloproteinase 7/metabolism , Sodium Chloride/pharmacology , Catalysis , Chromatography, High Pressure Liquid , Fluorescent Dyes/metabolism , Humans , Hydrolysis , Kinetics , Oligopeptides/metabolism , Protein Binding , Recombinant Proteins/metabolism , Temperature , Thermodynamics
20.
Biochim Biophys Acta ; 1481(2): 265-72, 2000 Sep 29.
Article in English | MEDLINE | ID: mdl-11018717

ABSTRACT

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.


Subject(s)
Cytochrome P-450 CYP1A1/metabolism , Microsomes/metabolism , Oxygen/metabolism , 7-Alkoxycoumarin O-Dealkylase/metabolism , Animals , Coumarins/metabolism , Cytochrome P-450 CYP1A1/genetics , Glycerol , Kinetics , Methanol , Microsomes/enzymology , Microsomes, Liver/enzymology , Rats , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Temperature
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