EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Effect of dietary proteins and carbohydrates on urinary and sympathoadrenal catecholamines

We previously observed that administration of tyrosine to rats or humans elevated urinary dopamine, norepinephrine and epinephrine levels. The present studies examine the effects on these urinary catecholamines of varying the ratio of protein to carbohydrate in the diets.Rats consumed diets containing 0, 18 or 40% protein (76, 58 and 36% carbohydrate respectively) for 8 days. The stress of consuming the protein-free food was associated with a 16% weight reduction, and with significantly lower serum, heart and brain tyrosine levels than those noted in rats eating the 18 or 40% protein diets. Absence of protein from the diet also decreased urinary levels of dopamine and DOPA but increased urinary norepinephrine and epinephrine, probably by increasing sympathoadrenal discharge; it also increased the excretion of DOPA in animals pretreated with carbidopa, a DOPA decarboxylase inhibitor. Carbidopa administration decreased urinary dopamine, norepinephrine and epinephrine as expected; however, among carbidopa-treated rats urinary norepinephrine and epinephrine concentrations were highest for animals consuming the protein-free diet, again suggesting enhanced release of stored catecholamines from sympathoadrenal cells. The changes in urinary catecholamines observed in animals eating the protein-free diet were similar to those seen in rats fasted for 5 days: dopamine levels fell sharply while norepinephrine and epinephrine increased.These data indicate that the effects of varying dietary protein and carbohydrate contents on dopamine secretion from peripheral structures differ from its effects on structures secreting the other two catecholamines. Protein consumption increases dopamine synthesis and release probably by making more of its precursor, tyrosine, available to peripheral dopamine-producing cells; it decreases urinary norepinephrine and epinephrine compared with that seen in protein-deprived animals, probably by diminishing the firing of sympathetic neurons and adrenal chromaffin cells.     

Triplet state properties of the methylmercury(II)-tyrosine complex

CH₃Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K_f defined by CH₃HgOH + HB

CH₃HgB + H₂O, we find log K_f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH₃Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm^?1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH₃Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T_z, where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.     

Hyperpigmented patches in the skin of the newt Notophthalmus viridescens

In the integument of the red-spotted newt there occasionally appear patches of skin which are at the same time melanistic and iridescent. Such hyperpigmented patches have been found on the back, on the tail and on the dorsal surface of both fore and hind limbs. Cytological examination of several such areas revealed the presence of large numbers of chromatophores distributed throughout the dermis. The majority of the chromatophores consisted of atypically large and dendritic melanophores, which contained typical pigment granules. The iridescence resulted from a high incidence of iridophores. Xanthophores also were found in considerable abundance. This extensive and apparently random intermingling of melanophores, iridophores and xanthophores in limited areas constitutes a striking exception to the usual distributional patterns of pigment cells in this animal.     

Biomass accumulation and shading effects of epiphytes on leaves of the seagrass, Heterozostera tasmanica, in Victoria,Australia

A method is described for estimating the rate of accumulation of epiphyte biomass on leaves of the seagrass, Heterozostera tasmanica (Martens ex Aschers.) den Hartog and for estimating the effect of epiphyte biomass on photosynthesis of the seagrass. Epiphyte biomass was determined by comparison of the weight per unit area of epiphyte-covered and epiphyte-free leaf blades. Epiphyte weight increased as age of the seagrass leaves increased. Linear regression on epiphyte biomass vs. leaf age estimated the rate of biomass accumulation. Rates varied from 5.7 to 104 μg epiphyte dry weight per cm² of leaf surface per day at three sites in Western Port and Port Phillip Bay, Victoria. Rates of accumulation of epiphyte biomass were generally higher during December through March (summer) than in May (autumn), August (winter) or October (Spring). Light attenuation by epiphytes increase linearly with biomass. The rate of biomass accumulation of epiphytes was compared with leaf growth rate, ambient photon flux density in H. tasmanica beds and the photosynthesis—photon flux density curve of H. tasmanica. This comparison demonstrated that epiphyte biomass can accumulate fast enough to shade H. tasmanica leaves and significantly reduce the time (to less than one half of the leaf life span) in which positive net photosynthesis of the leaf blade is possible.     

Seasonal variation in standing crop,density and leaf growth rate of the seagrass, Heterozostera tasmanica, in western Port and Port Phillip Bay,Victoria, Australia

Standing crop, density and leaf growth rate of Heterozostera tasmanica (Martens ex Aschers.) den Hartog along with light, temperature, nutrient and sediment characteristics were determined monthly for fifteen months at three study sites in Western Port and one site in Port Phillip Bay, Victoria, Australia. Erect vegetative stems of H. tasmanica were frequently branched, were present throughout the year and accounted for 25–60% of the above-sediment biomass, with the stem proportion higher during winter than summer. At three of the four sites there was a unimodal seasonal pattern in which minimum leaf standing crop (27–61 g dry wt. m^?2), density (600–2000 leaf cluster m^?2) and leaf productivity (0.34–0.77 g dry wt. m^?2 day^?1) generally occurred during winter (June–August) and maximum leaf standing crop (105–173 g dry wt. m^?2), density (2700–5000 leaf cluster m^?2) and leaf productivity (2.6–4.2 g dry wt. m^?2 day^?1) occurred during summer (December–February). A bimodal seasonal pattern with minimum standing crop and density during midsummer occurred at one site. This anomalous seasonal pattern may be due to exposure and desiccation stress during spring low tides. At the site receiving the lowest irradiance, standing crop, density and annual leaf production also were lowest, but length and width of leaves, shoot height and leaf growth rate per leaf cluster were the highest of the four study sites. On average, each leaf cluster at any one of the study sites produced 30–31 leaves per year with mean leaf turnover rates of 1.3–1.7% day^?1. Annual leaf production of H. tasmanica ranged from 410 to 640 g dry wt.m^?2 at the four sites.     

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

BACKGROUND: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes. METHODS: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV). RESULTS: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected. CONCLUSIONS: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.     

The copper-molybdenum antagonism in ruminants. I. The formation of thiomolybdates in animal rumen

The formation of thiomolybdates, MoO_xS_4?x^2?(x = 0, 1, 2, or 3), from molybdate and sulphide salts in aqueous media has been studied under conditions which simulate the fluid phase in the rumen. The influences of the sulphide:molybdenum ratio, pH and phosphate levels on the nature of the species formed were investigated. The thiomolybdates, in particular the MoS_4^2? ion, have been implicated as the active intermediates in the widespread molybdenum induced copper deficiency that affects ruminants. The results presented here show that, under physiological conditions, di- and trithiomolybdates will form more readily than tetrathiomolybdate.     

Spectroscopic studies on the copper(II) complexes of carnosine

Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 Å. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios     

A field study of the headbob displays of male green iguanas (Iguana iguana): Variation in form and context

Headbob displays play an important role in the social behaviour of male green iguanas (Iguana iguana). Four types of headbobs were identified, and the variability in form and context were determined from quantitative analyses of displays filmed in the field. The Roll is a conspicuous advertisement display. The Shudder, the most variable in form, occurred during close contact between males and females. The Roll-shudder is intermediate between the Roll and Shudder in its form and use. The Signature Bob, the most stereotyped display in form, punctuates much activity and is the most variable in contextual use. This display encodes species identity, and possibly individual identity. The conspicuous nature of the headbob displays was enhanced by the intensified colour of territorial males, and by the selection of highly visible display posts. This system of visual displays is viewed as the product of related sets of constraints, including the iguanas' physical capacities, the forest environment, and the social environment.     

The potent induction of intestinal heme oxygenase by the organotin compound, bis(tri-n-butyltin)oxide

Oral administration of bis(tri-n-butyltin)oxide, an important organotin biocidal agent, produces a substantial elevation in heme oxygenase activity when measured at 16 hours in rat small intestine. An apparent Km for hemin of 100 microM is the same in both control and the organotin-induced 9,000 X g supernatant preparations. Concomitant with elevated heme oxygenase activity there occurs a substantial reduction in benzo(a)pyrene hydroxylase activity (approximately 20% of controls) and cytochrome P-450 concentration (approximately 60% of controls). These perturbations of heme metabolism in intestinal epithelium of the rat define an important new toxicological effect of organotins and raise the possibility that concurrent oral ingestion of environmental pollutants can directly affect the cytochrome P-450-dependent metabolism of other chemicals in the intestine.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.     

Kinetics of Ni(II) and Co(II) monocomplex formation with O-phospho-dl-serine

The complexation reactions of O-phospho-DL-serine with Ni(II) or Co(II) were studied at 25°C and ionic strength 0.2 M (KNO₃) by temperature-jump. The observed rate constants for formation of the Ni²⁺ and Co⁺² monocomplexes were (1.32 ± 0.09) × 10⁵ and (1.73 ± 0.33) × 10⁷ M^?1 sec^?1, respectively. Complexation is postulated to involve formation of a monocoordinated steady state intermediate followed by rate-determining chelate ring closure.     

Radiorespirometry and metabolism of cultured cells attached to petri dishes

A radiorespirometer was constructed for continuous quantitation of ¹⁴CO₂ released from specifically labeled substrates by intact cultured cells attached to plastic petri dishes. An airtight chamber is created by sealing the petri dish with a specially designed cover inside a thermostated holder. Rapid equilibration of released ¹⁴CO₂ with a 5% $\text{CO}{}_2\text{95\%}$ air carrier gas is achieved by bubbling the carrier gas under the surface of the growth medium. Labeled CO₂ is removed from the carrier gas by trapping in an organic base and quantitated by liquid scintillation counting. Additions to or sampling of the growth medium may be performed during a run and the carrier gas may be modified to test the effects of anesthetics and different O₂ levels. The ability to continuously monitor ¹⁴CO₂ release can provide valuable information concerning the metabolic pathways of substrate oxidation which cannot be obtained from single ¹⁴CO₂ determinations. A capacity of 12 culture plates enormously increases the amount of data that can be collected in a given time. The use of liquid scintillation counting increases the sensitivity and resolution over the ion chamber and Geiger counter methods, and permits utilization of the procedure in a much wider range of laboratories. Data obtained for the oxidation of specifically ¹⁴C-labeled glucose and pyruvate by neonatal rat heart cells in culture, in both the presence and absence of oxygen, are provided as examples of the utility of the method.     

Inability of gestational hormones to account for the inhibitory effects of pregnancy plasmas on lymphocyte responses in vitro.

Plasma from pregnant women has a marked inhibitory effect on lymphocyte responses in vitro. While much evidence suggests that this is due to an immunologic mechanism, an apparent lack of specificity and the known suppressive effects of several hormones on immune function has led to speculation that the inhibitory effects could be due to increased concentrations of gestational hormones. We have investigated the effects of a wide range of concentrations of estrone, estradiol, estriol, progesterone, human chorionic gonadotropin (HCG), and hydrocortisone on lymphocyte responses to mitogens and allogeneic cells. None of these hormones were capable of inhibiting lymphocyte DNA synthesis even at concentrations several times the maximum physiologic plasma levels occurring during pregnancy. Very high, supraphysiologic concentrations were found to be inhibitory. In investigating the mechanism of the hormonal inhibition we found that if they were removed from the media at various times after initiation of culture, the estradiol, HCG, and to a lesser extent the hydrocortisone effects were all reversible. Estradiol and HCG differed from hydrocortisone in that the former were inhibitory only when added at the initiation of culture, whereas hydrocortisone was inhibitory even when added 24 hr later. In summary, while extremely high concentrations of gestational hormones are inhibitory, the quantities which occur physiologically in gestational plasmas are not able to suppress lymphocyte responses and thus cannot account for their inhibitory effects.