Short-term Hypoxia Increases Tyrosine Hydroxylase Immunoreactivity in Rat Carotid Body

Neurochemical and morphological changes in the carotid body are induced by chronic hypoxia, leading to regulation of ventilation. In this study, we examined the time courses of changes in immunohistochemical intensity for tyrosine hydroxylase (TH) and cellular volume of glomus cells in rats exposed to hypoxia (10% O₂) for up to 24 hr. Grayscale intensity for TH immunofluorescence was significantly increased in rats exposed to hypoxia for 12, 18, and 24 hr compared with control rats (p<0.05). The transectional area of glomus cells was not significantly different between experimental groups. The TH fluorescence intensity of the glomus cells exhibited a strong negative correlation with the transectional area in control rats (Spearman''s ρ = −0.70). This correlation coefficient decreased with exposure time, and it was lowest for the rats exposed to hypoxia for 18 hr (ρ = −0.18). The histogram of TH fluorescence intensity showed a single peak in control rats. The peaks were gradually shifted to the right and became less pronounced in hypoxia-exposed rats, suggesting that a hypoxia-induced increase in TH immunoreactivity occurred uniformly in glomus cells. In conclusion, this study demonstrates that short-term hypoxia induces an increase in TH protein expression in rat carotid body glomus cells. (J Histochem Cytochem 58:839–846, 2010)     

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.     

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.     

Predictive Power of a Body Shape Index for Development of Diabetes,Hypertension, and Dyslipidemia in Japanese Adults: A Retrospective Cohort Study

Background/Objectives

Recently, a body shape index (ABSI) was reported to predict all-cause mortality independently of body mass index (BMI) in Americans. This study aimed to evaluate whether ABSI is applicable to Japanese adults as a predictor for development of diabetes, hypertension, and dyslipidemia.

Subjects/Methods

We evaluated the predictive power of ABSI in a retrospective cohort study using annual health examination data from Chiba City Hall in Japan, for the period 2008 to 2012. Subjects included 37,581 without diabetes, 23,090 without hypertension, and 20,776 without dyslipidemia at baseline who were monitored for disease incidence for 4 years. We examined the associations of standardized ABSI, BMI, and waist circumference (WC) at baseline with disease incidence by logistic regression analyses. Furthermore, we conducted a case-matched study using the propensity score matching method.

Results

Elevated BMI, WC, and ABSI increased the risks of diabetes and dyslipidemia [BMI-diabetes: odds ratio (OR) = 1.26, 95% confidence interval (95%CI) = 1.20−1.32; BMI-dyslipidemia: OR = 1.15, 95%CI = 1.12−1.19; WC-diabetes: OR = 1.24, 95%CI = 1.18−1.31; WC-dyslipidemia: OR = 1.15, 95%CI = 1.11−1.19; ABSI-diabetes: OR = 1.06, 95%CI = 1.01−1.11; ABSI-dyslipidemia: OR = 1.04, 95%CI = 1.01−1.07]. Elevated BMI and WC, but not higher ABSI, also increased the risk of hypertension [BMI: OR = 1.32, 95%CI = 1.27−1.37; WC: OR = 1.22, 95%CI = 1.18−1.26; ABSI: OR = 1.00, 95%CI = 0.97−1.02]. Areas under the curve (AUCs) in regression models with ABSI were significantly smaller than in models with BMI or WC for all three diseases. In case-matched subgroups, the power of ABSI was weaker than that of BMI and WC for predicting the incidence of diabetes, hypertension, and dyslipidemia.

Conclusions

Compared with BMI or WC, ABSI was not a better predictor of diabetes, hypertension, and dyslipidemia in Japanese adults.     

Rheological characteristics of desialylated erythrocytes in relation to fibrinogen-induced aggregation

The effect of fibrinogen and sialic acid content of erythrocytes on the aggregation of erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (1) The electrophoretic mobility of erythrocytes was proportional to the sialic acid content of erythrocytes (the surface potential of erythrocytes could be expressed by the sialic acid content). (2) The aggregation of erythrocytes was accelerated by increasing fibrinogen concentration in the medium (due to the increased bridging force among erythrocytes) or by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes). (3) An empirical equation expressing the velocity of aggregate formation (ν, in μm²/min) by the concentration of fibrinogen ($\text{F}$, in g/dl) and the sialic acid content ($\text{S}$, in μmol/ml red blood cells), log $\text{ν = ?0.065 F}{}^{\mathrm{?1.2}}\text{S + 2.2 F}{}^{0.35}$, was deduced. (4) The contribution of the bridging force of fibrinogen to the erythrocyte aggregation was much greater than that of the electrostatic repulsive force produced by sialic acid on the surface of erythrocytes.     

Two Distinct Pathways for Targeting Proteins from the Cytoplasm to the Vacuole/Lysosome

Stress conditions lead to a variety of physiological responses at the cellular level. Autophagy is an essential process used by animal, plant, and fungal cells that allows for both recycling of macromolecular constituents under conditions of nutrient limitation and remodeling the intracellular structure for cell differentiation. To elucidate the molecular basis of autophagic protein transport to the vacuole/lysosome, we have undertaken a morphological and biochemical analysis of this pathway in yeast.     

Immunochemical characteristics of a novel cell death receptor and a decoy receptor on granulosa cells of porcine ovarian follicles

Previously, we prepared an IgM monoclonal antibody(PFG-1) which specifically recognized a cell-membraneglycoprotein (PFG-1 antigen; 55 kD, pI 5.9),immunohistochemically reacted with granulosa cells ofhealthy follicles but not of atretic follicles, andinduced granulosa cell apoptosis. In the presentstudy, an IgM monoclonal antibody (PFG-3) capable ofinducing granulosa cell apoptosis and an IgGmonoclonal antibody (PFG-4) not capable of inducingapoptosis were produced against granulosa cellsprepared from healthy antral follicles of porcineovaries. Two-dimensional Western blotting analysisrevealed that PFG-3 specifically recognized twocell-membrane proteins (named PFG-3-1 andPFG-3-2/PFG-1 antigens; 42 kD, pI 5.2 and 55 kD, pI5.9, respectively) of healthy granulosa cells, andthat PFG-4 recognized the same two cell-membraneproteins. In atretic granulosa cells, PFG-3-2/PFG-1antigen disappeared. Immunochemical reactions of theseantibodies were only detected in follicular granulosacells but not any other ovarian tissues or organs.PFG-3 and PFG-4 immunohistochemically reacted withgranulosa cells of healthy and atretic follicles. Whenthe isolated granulosa cells prepared from healthyfollicles were cultured in medium containing PFG-3,the cells underwent apoptosis, and co-incubation withPFG-4 inhibited PFG-3-inducible apoptosis. Theseobservations suggested that PFG-3-2/PFG-1 antigen isa novel cell death receptor which is different fromthe apoptosis-mediating receptors (Fas/Apo-1/CD95 orTNF receptor), and that PFG-3-1 antigen may act as adecoy receptor and inhibit apoptotic signal transmission.     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

A Genomic Screen for Yeast Mutants Defective in Selective Mitochondria Autophagy

Mitophagy is the process of selective mitochondrial degradation via autophagy, which has an important role in mitochondrial quality control. Very little is known, however, about the molecular mechanism of mitophagy. A genome-wide yeast mutant screen for mitophagy-defective strains identified 32 mutants with a block in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We further characterized one of these mutants, ylr356wΔ that corresponds to a gene whose function has not been identified. YLR356W is a mitophagy-specific gene that was not required for other types of selective autophagy or macroautophagy. The deletion of YLR356W partially inhibited mitophagy during starvation, whereas there was an almost complete inhibition at post-log phase. Accordingly, we have named this gene ATG33. The new mutants identified in this analysis will provide a useful foundation for researchers interested in the study of mitochondrial homeostasis and quality control.