The relationship between IMPS-measured stress score and intraocular pressure among public school workers

The aim of this study was to examine the relationship between psychosocial stress and intraocular pressure among apparently healthy subjects. Psychosocial stress among 1,461 public school workers (883 men and 578 women) was measured using the inventory to measure psychosocial stress (IMPS) and intraocular pressure was measured using a non-contact tonometer (Topcon CT-90). After controlling for the effects of likely confounding variables such as age, body mass index (BMI), glycosylated hemoglobin, systolic blood pressure, alcohol consumption, smoking status, and exercise, partial correlations and hierarchical multiple regression analysis were performed in order to test the hypothesis that IMPS-measured stress score was associated with intraocular pressure. IMPS-measured stress score was found to correlate positively with intraocular pressure in women after controlling for the effects of confounding variables, whereas this relationship was not found in men. Hierarchical multiple regression analysis indicated that IMPS-measured stress score was positively associated with intraocular pressure in women independent of confounding variables, but not in men. Perturbations of the hypothalamic-pituitary-adrenal (HPA) axis associated with stress are considered to be partly responsible for an increase in intraocular pressure among people suffering from psychosocial stress. Further research is needed to elucidate the relationship between this stress-associated increase in intraocular pressure and open-angle glaucoma.     

Cannabinoids act as necrosis-inducing factors in Cannabis sativa

Cannabis sativa is well known to produce unique secondary metabolites called cannabinoids. We recently discovered that Cannabis leaves induce cell death by secreting tetrahydrocannabinolic acid (THCA) into leaf tissues. Examinations using isolated Cannabis mitochondria demonstrated that THCA causes mitochondrial permeability transition (MPT) though opening of MPT pores, resulting in mitochondrial dysfunction (the important feature of necrosis). Although Ca²⁺ is known to cause opening of animal MPT pores, THCA directly opened Cannabis MPT pores in the absence of Ca²⁺. Based on these results, we conclude that THCA has the ability to induce necrosis though MPT in Cannabis leaves, independently of Ca²⁺. We confirmed that other cannabinoids (cannabidiolic acid and cannabigerolic acid) also have MPT-inducing activity similar to that of THCA. Moreover, mitochondria of plants which do not produce cannabinoids were shown to induce MPT by THCA treatment, thus suggesting that many higher plants may have systems to cause THCA-dependent necrosis.Key words: cannabinoid, Cannabis sativa, cylophilin D, mitochondrial permeability transition, necrosisCannabis sativa produces unique secondary metabolites consisting of alkylresorcinol and monoterpene groups.¹ These metabolites called cannabinoids are well known to show a variety of interesting pharmacological activities including psychoactive effect and analgesic effect. Therefore, cannabinoids have attracted a great deal of attention, whereas why C. sativa produces such metabolites has long remained unclear. However, we have recently obtained evidences indicating the physiological function of THCA in Cannabis leaves.²We discovered that THCA is stored in capitate-sessile glands on Cannabis leaves and that secretion of this cannabinoid into leaf tissues causes cell death. When the properties of THCA were examined using cultured Cannabis cells, this cannabinoid induced plasmamembrane shrinkage and DNA degradation. These responses are regarded as the features of apoptotic cells, but were not suppressed by apoptosis inhibitors. In contrast, the necrosis inhibitor cyclosporine A significantly inhibited both plasmamembrane shrinkage and DNA degradation in Cannabis cells. Therefore, we assumed that THCA induces necrotic cell death in Cannabis cells and leaves.Necrosis in plants and animals is usually triggered by MPT though opening of MPT pores.^3,4 MPT is known to cause mitochondrial dysfunction by mitochondrial swelling and loss of mitochondrial membrane potential (ΔΨm),^5,6 and we also confirmed that THCA induces mitochondrial swelling and ΔΨm reduction in mitochondria isolated from Cannabis cells and that pretreatment with cyclosporine A inhibits both responses. Based on these evidences, we concluded that THCA has the activity to induce MPT-dependent necrosis.As described above, MPT pores play an important role in necrosis induction, whereas the mechanism of their opening in higher plants has not been fully understood. However, binding of cyclophilin D (a protein present in mitochondrial matrix) to MPT pores is shown to be essential for their opening in plants as well as animal.^7–9 In animal mitochondria, Ca²⁺ mediates this binding reaction, leading to opening of MPT pores. Wheat mitochondria are also shown to undergo swelling through opening of MPT pores in response to Ca²⁺,⁹ whereas MPT pores of oats,¹⁰ Arabidopsis thaliana¹¹ and C. sativa² do not open by Ca²⁺ treatment. In contrast, THCA catalyzed opening of Cannabis MPT pores in the absence of Ca²⁺, suggesting that THCA directly mediates binding of cyclophilin D to MPT pores (Fig. 1). In addition, we have now confirmed that THCA causes dysfunction though MPT in mitochondria of plants (rice, soybean, A. thaliana and Scutellaria baicalensis) lacking cannabinoid-producing ability (data not shown). Therefore, many higher plants may have the systems to induce THCA-dependent necrosis.Open in a separate windowFigure 1A model depicting the opening mechanism of MPT pores in mitochondria. CYD, cyclophilin D; CN, cannabinoid.Furthermore, we investigated whether other cannabinoids and their related compounds can mediate MPT in Cannabis mitochondria. When the MPT-inducing activity of each sample was measured by monitoring both ΔΨm reduction (Fig. 2) and mitochondrial swelling (data not shown), we confirmed that cannabinoids tested here (cannabidiolic acid and cannabigerolic acid) possess the activities similar to those of THCA. On the other hand, olivetolic acid (the akylresorcinol moiety of cannabinoid) and geraniol (the monoterpene moiety of cannabigerolic acid) showed neither ΔΨm reduction nor mitochondrial swelling (Fig. 2). These results suggested that the structures (cannabinoid skeleton) where monoterpene and olivetolic acid are coupled to each other seem essential for opening of MPT pores. Therefore, we assumed that plant cyclophilin D and MPT pores have the cannabinoid-binding site.Open in a separate windowFigure 2Change of ΔΨm by treatment with various compounds (A) and their chemical structures (B). The isolated mitochondria were stained with the ΔΨm-indicating reagent (tetramethylrhodamine methylester, TMRM) and then incubated with 200 µM of each compound for 60 min. The intensity of TMRM fluorescence was measured using a fluorescence microplate reader. A decrease of the fluorescence intensity indicates ΔΨm reduction. CBDA, cannabidiolic acid; CBGA, cannabigerolic acid; OLA, olivetolic acid.Plant cell death is shown to participate in important physiological responses such as leaf senescence, somatic embryogenesis and defense against microbial pathogens.^12,13 Based on its induction mechanism, plant cell death is largely classified into apoptosis and necrosis. Although the molecular mechanism of apoptosis has been extensively investigated, there is little precise information on plant necrosis. However, our study would provide important insight into necrosis-inducing mechanisms in higher plants.     

Pancreatic islet blood flow in conscious rats during hyperglycemia and hypoglycemia

Anesthesia affects general hemodynamics and regulation of organ perfusion. We used colored microspheres to measure pancreatic islet blood flow in conscious rats at two time points, during either hyperglycemia or hypoglycemia. This method, using black and green microspheres, was validated by comparison with previous microsphere experiments and by lack of effect of a nonmetabolizable glucose analog, 3-O-methylglucose, on islet perfusion. Basal and glucose-stimulated islet blood flow levels were similar in pentobarbital sodium-anesthetized and conscious rats. However, the basal distribution of pancreatic blood flow was altered by anesthesia (fractional islet blood flow 5.8 +/- 0.4% in conscious rats, 7.9 +/- 0.8% in pentobarbital-anesthetized rats, P < 0.05). Insulin-induced hypoglycemia significantly increased whole pancreatic blood flow in conscious rats, whereas islet blood flow remained unchanged and fractional islet blood flow was decreased (5.8 +/- 0.5% in the basal state, 4.2 +/- 0.4% during hypoglycemia, P < 0.001). Methylatropine pretreatment significantly increased islet blood flow during hypoglycemia by 181%. This result suggests that prevention of hypoglycemia-induced increase in islet perfusion may be mediated, at least in part, by a cholinergic, vagal muscarinic mechanism.     

Life history elasticity and the population-level effect of p-nonylphenol on Daphnia galeata

In order to evaluate population-level effects of p-nonylphenol on a cladoceran zooplankton (Daphnia galeata), the chronic effects on survival and reproduction were estimated with partial life table tests, which examined responses in life history characters until 3 weeks after birth. The observed responses in survival and reproduction were converted to reductions of the intrinsic rate of natural increase r. The population level EC₅₀, which is defined as the exposure concentration that reduces r by 50%, was estimated as 16.1 g l^–1. In order to examine the extent to which the population-level effect in terms of r is influenced by extra mortality in nature, which is induced by predation, starvation, etc., sensitivity (elasticity) measures of the intrinsic rate of natural increase to reductions in age-specific survival and reproduction were calculated under hypothetical predation schemes. The sensitivities of the intrinsic rate to changes in survival and reproduction invariably decline rapidly after the onset of reproduction irrespective of predation schemes. This implies that partial life cycle tests until 21 days after birth can provide reliable estimates of the population-level effects.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Impaired lymphopoiesis and altered B cell subpopulations in mice overexpressing Lnk adaptor protein

Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk(-/-) mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk(-/-) hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.     

Estrogen activation of the nuclear orphan receptor CAR (constitutive active receptor) in induction of the mouse Cyp2b10 gene

The nuclear orphan receptor CAR (constitutively active receptor or constitutive androstane receptor) can be activated in response to xenochemical exposure, such as activation by phenobarbital of a response element called NR1 found in the CYP2B gene. Here various steroids were screened for potential endogenous chemicals that may activate CAR, using the NR1 enhancer and Cyp2b10 induction in transfected HepG2 cell and/or in mouse primary hepatocytes as the experimental criteria. 17beta-Estradiol and estrone activated NR1, whereas estriol, estetrol, estradiol sulfate, and the synthetic estrogen diethylstilbestrol did not. On the other hand, progesterone and androgens repressed NR1 activity in HepG2 cells, and the repressed NR1 activity was fully restored by estradiol. Moreover, estrogen treatment elicited nuclear accumulation of CAR in the mouse livers, as well as primary hepatocytes, and induced the endogenous Cyp2b10 gene. Ovariectomy did not affect either the basal or induced level of CAR in the nucleus of the female livers, while castration slightly increased the basal and greatly increased the induced levels in the liver nucleus of male mice. Thus, endogenous estrogen appears not to regulate CAR in female mice, whereas endogenous androgen may be the repressive factor in male mice. Estrogen at pharmacological levels is an effective activator of CAR in both female and male mice, suggesting a biological and/or toxicological role of this receptor in estrogen metabolism. In addition to mouse CAR, estrogens activated rat CAR, whereas human CAR did not respond well to the estrogens under the experimental conditions.