Spectral sensitivity of photoreceptors mediating phase-shifts of circadian rhythms in retinally degenerate CBA/J (rd/rd) and normal CBA/N (+/+) mice

Light-dark cycles are the most important time cue for the circadian system to entrain the endogenous circadian clock to the environmental 24 h cycle. Although photic entrainment of circadian rhythms is mediated by the eye in mammals, photoreceptors implicated in circadian photoreception remain unknown. In our previous study, retinally degenerate CBA/J (rd/rd) mice were found to have lower circadian photo-sensitivity for phase-shifting the locomotor activity rhythms than normal CBA/N(+/+) mice. In the present study, the spectral sensitivity for phase-shifting the rhythms was examined in order to characterize the photopigments involved in circadian photoreception of these mice. The spectral sensitivity of CBA/J-rd/rd mice clearly fitted to the Dartnall nomogram for a retinal₁-based pigment with a maximum at 480 nm, while the best fitted nomogram had a maximum at 500 nm in CBA/N- +/+ mice. These results suggest that circadian photopigments involved in CBA/J-rd/rd and CBA/N- +/+ mice may be different.     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

The 21-aminosteroid U-74389F increases the number of glial fibrillary acidic protein-expressing astrocytes in the spinal cord of control and wobbler mice

Summary 1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Givenin vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [³H]dexamethasone binding when addedin vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Selenium,zinc, and thyroid hormones in healthy subjects

Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T₃ production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral conversion of T₄ to T₃ with a lower T₃/T₄ ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T₃/T₄ ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation between the T₃/T₄ ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as compared with younger subjects. We concluded that reduced peripheral T₄ conversion is related to impaired Se status in the elderly.     

Halcyon days with Bill Arnold

The circumstances that led to the discovery that plants luminesce after they are illuminated are described, as are other discoveries that would not have been possible were it not for the fortuitous association I had with my dear and most admirable friend, W.A. Arnold, to whom this special issue is dedicated.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Soil nitrogen dynamics along a gradient of long-term soil development in a Hawaiian wet montane rainforest

I analyzed the rates of net N mineralization and nitrification of soils from seven sites in a Hawaiian wet montane forest. The sites differ in age, ranging from 400 to 4,100,000 yr, but are comparable in other variables (all at 1200 miasl with 4000 mm or more mean annual rainfall), and the chronosequence simulated a development of soils from basaltic lava. Soils were incubated for 20 days at 17.5 °C, which is nearly equivalent to a mean field air temperature of the sites, and at an elevated temperature of 25.5 °C under three treatments: 1) field-wet without amendments, 2) air dried to a permanent wilting point, and 3) fertilized with phosphate (NaH₂PO₄) at the rate of 50 g P per g dry soil. Both mineralization and nitrification rates varied significantly among the sites at the field temperature (p<.00001). Fractions of the mineralized organic matter (indexed by the N produced per g organic C) increased sharply from the youngest to the 5000-yr site before declining abruptly to a near constant value from the 9000 to the 1,400,000-yr sites. Total organic C in the top soils (<15 cm deep) increased almost linearly with age across the sites. Consequently, net NH₄- and NO₃-N produced on an area basis (g m^-2 20 d^-1) increased sharply from 0.2 in the youngest site to 1.2 in the 5000-yr site, then both became depressed once but steadily increased again. The fraction of organic matter mineralized, and the net N turnover rates were outstandingly high in the oldest site where a large amount of organic matter was observed; the topsoil organic matter which was used in this analysis appeared to be highly labile, whereas the subsurface organic matter could be relatively recalcitrant. As suggested by earlier workers, the initial increase in N turnover seemed to correspond to the increasing quantity of N in the soils through atmospheric deposition and biological fixation. The later decline in fraction of organic matter mineralized seemed to relate to increasing soil C/N ratios, increasingly recalcitrant organic matter, and poorer soil drainage with age. The elevated temperature treatment produced significantly higher amounts of N mineralization, except for the youngest site where N was most limiting, and for two sites where soil waterlogging might be severe. P fertilization invariably resulted in slower N turnovers, suggesting that soil microbes responded to added P causing N immobilization. The youngest site did not significantly respond to added P. The magnitude of immobilization was higher in older than in younger soils, suggesting that P more strongly limits microbial populations in the older soils.     

Zooplankton community response to reservoir aging

Changes in zooplankton diversity and density in response to reservoir aging in Pawnee Reservoir were investigated. Zooplankton samples collected from April 1992 through April 1993, were compared to a similar study conducted after initial impoundment by Helzer (1971), in 1968–1970. Since this initial study, increases in turbidity and resulting changes in biotic interactions significantly altered the zooplankton community. A significant increase in total zooplankton density and a decrease in species richness were observed between study periods. Density increased from 24.6 to 95.4 individuals L^–1, while the number of taxa declined from fourteen to ten. During this time period, Cyclops vernalis became the dominant zooplankter in the reservoir. The density of this predatory copepod increased significantly, from 0.1 l^–1 in 1968–1970, to 44.3 l^–1 in 1992–1993, which accounted for most of the increase in total zooplankton density. Though a greater spring maximum of another dominant, Bosmina spp. was found during the 1992–1993 study period, the annual density of this cladoceran was not significantly different between study periods. Similar trends for Daphnia ambigua and D. parvula were also observed, as greater spring maxima levels were attained, however overall annual densities were not significantly different. The dominance of C. vernalis (46% of annual density) and Bosmina spp. (33%), indicate that these two zooplankters were tolerant of changes in physical conditions resulting from reservoir aging and biotic interactions that followed in the reservoir during the 22 years between study periods.