Transition assignments in the ultraviolet-visible absorption and magnetic circular dichroism spectra of phthalocyanines.
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Journal Article↗
Biomedical subjects
M J Stillman
Publications and source records attributed to M J Stillman.
Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein.
The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.
Animals↗
The bedside confusion scale: development of a portable bedside test for confusion and its application to the palliative medicine population.
BACKGROUND: Clinical tests for confusion in medically ill patients are frequently burdensome and difficult to use. Available tests lack portability and tend to be shunned in clinical practice by physicians. OBJECTIVE: To develop a simple, sensitive bedside test for confusion. DESIGN: Prospective comparison study. SETTING: An in-patient palliative medicine unit in a large urban hospital. PATIENTS: Thirty-one consecutive patients admitted to the unit. INTERVENTION: None. MEASUREMENTS: A 2-minute screening test, the Bedside Confusion Scale (BCS), which utilizes an observation of level of consciousness at the time of clinical interaction, followed by a timed task of attention, was administered to 31 consecutively admitted patients. The results were compared to a previously validated test, the Confusion Assessment Method (CAM). The BCS and the CAM were scored in standardized fashion and results of the two populations compared. Demographic and clinical characteristics of the patient population, along with the Karnofsky performance scores (KPS) and neurological findings were registered. RESULTS: Using the CAM as the reference standard, the sensitivity of the BCS was 100%. Worsening KPS and more abnormalities on neurological examination were seen across normal (BCS = 0), borderline (BCS = 1), and abnormal (BCS >/= 2) groups (p > 0.01, trend test). CONCLUSIONS: In an in-patient palliative medicine population, the BCS correlates with the previously validated CAM and exhibits high sensitivity, an essential quality of a useful screening test.
Journal Article↗
Differences in physician access patterns to hospice care.
Few issues in health care have recently generated as much discussion as the two seemingly unrelated topics of out-of-hospital health care financing and compassionate care of patients at the end of life. These two topics meet where health care costs cross paths with the economic viability of hospice and palliative medicine. In this study, we evaluated 101 admissions to a large Medicare-certified hospice in the last quarter of 1995 to assess factors associated with timing of referral to hospice. Mean length of stay in hospice was 55 days; median was 23 days. The majority of patients had cancer diagnoses (74%). Contrary to our hypothesis, there was no statistically significant difference in mean patient lengths of stay between oncologist-referred and nononcologist-referred patients. However, when we compared patient lengths of stay lasting less than--versus longer than--30 days, more patients referred by nononcologists were in hospice longer than 30 days (chi 2 = 3.92, P < 0.05). With further evaluation, this difference was attributable to longer stays by patients covered by the Medicine hospice benefit, by those with noncancer diagnoses, and by those who were older. More of these patients were referred by nononcologists. The difference in referral patterns between oncologists and nononcologists disappeared when only cancer patients were considered. Consistent with initial hypotheses, caregivers of patients with shorter lengths of stay were significantly less satisfied with hospice care (t = -4.06, P < 0.001). These results suggest that health care benefits and other patient-specific issues influence timing of hospice referral rather than simply preferences by types of physicians. The impact on Medicare expenditures and hospice viability is discussed.
Adolescent↗
Spatial memory under acute cold and restraint stress.
This study examined spatial memory as measured by radial arm maze (RAM) performance after exposure to two stress conditions and a normothermic-unrestrained control condition. Male Fischer 344 rats were trained on the win-shift RAM procedure for 7 days, by which time they achieved asymptotic performance. The next day, rats in the two stress groups were exposed to 15 min of restraint in either 37 degrees C water (normothermic-restraint) or in 20 degrees C water (cold-restraint). Rats were then allowed 40 min in a dry cage before being tested in the RAM. Performance was measured using the following dependent variables: number of correct out of the first eight choices, total number of choices, and time per choice. There were statistically significant effects of stress on all these variables; performance decrements were observed in both stress conditions relative to the normothermic-unrestrained condition. Normothermic-restrained rats displayed less impairment than cold-restrained rats on the stress day. Performance of normothermic-restrained rats returned to baseline levels the day after stress, whereas performance for the cold-restrained rats typically did not. This study demonstrates that: 1) restraint and cold stress impair performance on a memory task; and 2) impairment extent is related to stress severity. One of the mechanisms responsible for the observed behavioral deficits under cold stress may involve altered cholinergic function, because we previously demonstrated that hippocampal acetylcholine levels also decrease in relation to the severity of cold stress.
Animals↗
Incorporation of copper into the yeast Saccharomyces cerevisiae. Identification of Cu(I)--metallothionein in intact yeast cells.
Copper is an essential metal ion to many living organisms, including mammals, as it mediates a wide variety of important biochemical processes. At elevated concentrations, copper is extremely toxic to host cells. This paradoxical nature of copper has necessitated a highly regulated procedure for its cellular accumulation, transport, and excretion. One important group of proteins involved in eukaryotic copper speciation is the protein metallothionein. Luminescence microscopy data, emission, and circular dichroism spectral data are reported as copper is incorporated into metallothionein by the yeast Saccharomyces cerevisiae. These techniques provide information on the mechanism of copper uptake by S. cerevisiae. A two-stage kinetic mechanism for the uptake of copper from the growth medium by the yeast cells is observed. The first stage displays an uptake rate that is dependent on the initial copper concentration of the growth medium, and lasts for approximately 6 h. The second stage has a slower rate of copper uptake than the first, but the kinetics are independent of the initial copper concentration. Emission spectra recorded directly from the intact yeast cells (at 77 K) show that the cellular incorporation of copper proceeds via several species, eventually leading to storage of the copper in the form of Cu-metallothionein. The photomicrographs of yeast cells grown in a copper-containing medium clearly show an orange luminescence, indicating the formation of a Cu(I)-thiolate species. The identification of this species as copper-metallothionein was confirmed by measurement of the circular dichroism and emission properties following excretion and isolation of the copper-containing protein from the yeast cells. Analysis of the emission spectrum from S. cerevisiae Cu-metallothionein at 77 K reveals two emission bands, centered at 570 and 700 nm. The high-energy emission band exhibits a two-component decay, with excited state lifetimes of 4.70 and 48.5 microseconds. The low-energy emission exhibits one major decay component with a lifetime of 1.13 microseconds. A high-molecular-weight, copper-containing species is also isolated from the yeast cells and is characterized spectroscopically.
Carrier Proteins↗
Comparison of the structures of the metal-thiolate binding site in Zn(II)-, Cd(II)-, and Hg(II)-metallothioneins using molecular modeling techniques.
The first fully energy-minimized structures for a series of structurally related metal complexes of the important mammalian metal binding protein metallothionein are described. The structures were calculated based on structural information obtained from existing spectroscopic and crystallographic data, and minimized using molecular mechanics (MM2) techniques. A two domain structure, with stoichiometries of M(II)3-(Scys)9 and M(II)4-(Scys)11 where M = zinc(II), cadmium(II), and mercury(II), was assembled and minimized. The resultant three-dimensional structure closely resembled that of rat liver Cd5Zn2-MT 1 obtained by analysis of x-ray diffraction data [A. H. Robbins, D.E. McRee, M. Williamson, S. A. Collett, N. H. Xuong, W. F. Furey, B. C. Wang and C. D. Stout, J. Mol. Biol. 221, 1269-1293 (1991)]. Minimized structures for Zn7-MT, Cd7-MT, and Hg7-MT are reported. Deep crevices that expose the metal-thiolate clusters are seen in each structure. However, for the mercury-containing protein, much of the mercury-thiolate structure is visible and it is proposed that this provides access for extensive interaction between solvent water molecules and the mercury(II), resulting in the observed distortion away from tetrahedral geometry for Hg7-MT. Volume calculations are reported for the protein metallated with 7 Zn(II), Cd(II), or Hg(II). A series of structural changes calculated for the step-wise isomorphous replacement of Zn(II) by Cd(II) and Hg(II) in the Zn4S11 alpha domain are shown.
Amino Acid Sequence↗
Effects of M2 antagonists on in vivo hippocampal acetylcholine levels.
There is evidence that muscarinic receptors of the M2 subtype are presynaptic autoreceptors that modify the release of acetylcholine (ACh) through a negative feedback mechanism. Blocking these receptors by selective antagonists may therefore lead to increased ACh release. This in vivo microdialysis study examined the effects of three M2 antagonists, AF-DX 116, AF-DX 384, and AQ-RA 741, on hippocampal cholinergic neurotransmission. Drug (2, 4, 8, or 16 microM) or vehicle (Ringer's solution) was perfused via a microdialysis probe into the CA1 hippocampal region of conscious male Fischer 344 rats. Levels of ACh and choline were assessed by HPLC-EC. When the dose was expressed in K1 multiples, all drugs (except AQ-RA 741 at the two highest concentrations) were found to be on the same linear dose-response curve. Choline levels were not affected by drug administration. All three compounds elevated ACh levels in a similar K1-normalized dose-response fashion, strongly supporting the concept that the proposed presynaptic mechanism of action is indeed based on the same M2 receptor. Such elevations of ACh may not only improve performance on memory tasks, but may also have therapeutic advantages in conditions of cholinergic hypofunction, such as Alzheimer's disease.
Acetylcholine↗
Morphological alterations in the hippocampus following hypobaric hypoxia.
1. The morphological consequences of hypobaric hypoxia, exposure to reduced pressure atmospheres, were examined in the hippocampus of male Fischer 344 rats. Severe chronic hypoxia can produce permanent neuronal damage with hippocampal structures being especially vulnerable. 2. Hippocampal morphology was studied using histological observations after a 4 day exposure to sea level, 3500 m, or 6400 m. Two groups tested at 6400 m were sacrificed at different intervals following exposure, 72 and 144 h, to examine the effect of post-exposure time on neuronal damage. 3. Histological damage was observed in rats' brains following exposure to altitude, with cell degeneration and death increasing as altitude increased. In addition, it was found that the longer the time following exposure before sacrifice, the more noticeable the damage, suggesting delayed neurotoxicity. Increases in the number of damaged cells following altitude were significant for the CA3 region of one 6400 m group; however, other differences did not reach statistical significance. Rats exposed to altitude for 4 days ate less and lost significantly more weight than did animals at sea level. 4. It appears that 4 days of exposure to altitudes less than or equal to 6400 m does produce changes in the CA3 subfield, but the damage is different than that seen with other models of non-transient ischemia.
Altitude Sickness↗
Determination of the hydroxyapatite-nucleating region of bone sialoprotein.
Bone sialoprotein (BSP) was shown to be a potent nucleator of hydroxyapatite (HA) in a steady-state agarose gel system (Hunter and Goldberg, 1993, PNAS 90: 8562). Nucleation of HA was also demonstrated with the homopolymer poly-glutamic acid but not with poly-aspartic acid or osteopontin. Since BSP contains contiguous sequences of glutamic acid, it is reasonable to suggest that the HA-nucleating activity of BSP resides within these regions. Purified porcine BSP was treated with trypsin and digests fractionated by gel filtration. In addition to small peptides (P3-5), two peptides of 38 kDa (P1) and 25 kDA (P2) were recovered, and after characterization assigned to the regions within BSP encompassing residues 133-272 (P1) and 42-125 (P2). Each of these peptides contained one of the two glutamic acid-rich regions of porcine BSP. In the steady-state agarose gel system, BSP, P1 and P2 induced HA formation, whereas the pooled small BSP-derived peptides (P3-5) did not. Analysis by circular dichroism spectroscopy revealed that the homopolymer poly-L-glutamic acid assumes a helical structure, while poly-L-aspartic acid does not. These findings suggest that the nucleating activity does not require intact molecules, that the nucleation of HA and BSP appears to require glutamic acid-rich sequences in a helical conformation and that there are two domains in porcine BSP that are each capable of nucleating HA.
Animals↗
Copper binding to rabbit liver metallothionein. Formation of a continuum of copper(I)-thiolate stoichiometric species.
Circular dichroism and ultraviolet absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(I)-thiolate binding clusters in rabbit liver metallothionein during addition of Cu+ to aqueous solutions of Zn7-metallothionein 2 and Cd5Zn2-metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the alpha and beta domains. Spectral data which probe the distribution of Cu(I) between the two binding domains clearly show that both the site of binding (alpha or beta), and the structures of the specific metal-thiolate clusters formed, are dependent on temperature and on the nature of the starting protein (either Zn7-metallothionein or Cd5Zn2-metallothionein). CD spectra acquired during the addition of Cu+ to Zn7-metallothionein show that Cu+ replace the bound Zn(II) in a domain-distributed manner with complete removal of the Zn(II) after addition of 12 Cu+. Spectral and metal analyses prove that a series of Cu(I)-metallothionein species are formed by a non-cooperative metal-binding mechanism with a continuum of Cu(I):metallothionein stoichiometries. Observation of a series of spectral saturation points signal the formation of distinct optically active Cu(I)-thiolate structures for the Cu9Zn2-metallothionein, Cu12-metallothionein, and the Cu15-metallothionein species. These data very clearly show that for Cu(I) binding to Zn7-metallothionein, there are several key Cu(I):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu12-metallothionein species are defined by bands located at 255(+) nm and 280(-) nm. Interpretation of the changes in the CD and ultraviolet absorption spectral data recorded between 3 degrees C and 52 degrees C as Cu+ is added to Zn-metallothionein show that copper-thiolate cluster formation is strongly temperature dependent. These changes in spectral properties are interpreted in terms of kinetic versus thermodynamic control of the metal-binding pathways as Cu+ binds to the protein. At low temperatures (3 degrees C and 10 degrees C) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper-thiolate structures until formation of Cu12-metallothionein. At higher temperatures (> 30 degrees C) the activation barrier is overcome, allowing formation of new Cu(I)-thiolate clusters with unique spectral properties, especially at the Cu9Zn2-metallothionein point. The CD spectra also show that a Cu15-metallothionein species with a well-defined, three-dimensional structure forms at all temperatures, characterized by a band near 335 nm, indicating the presence of diagonal Cu(I).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Analysis of the optical absorption and magnetic-circular-dichroism spectra of peanut peroxidase: electronic structure of a peroxidase with biochemical properties similar to those of horseradish peroxidase.
The electronic structures of the cationic isoenzyme of peanut peroxidase, horseradish peroxidase (isoenzyme C) and bovine liver catalase are compared through analysis of their optical absorption and magnetic c.d. (m.c.d.) spectral properties. The spectral data for the native resting states and compounds I and II of peanut peroxidase (PeP) are reported. The absorption and m.c.d. data for the native PeP exhibit bands characteristic of the high-spin ferric haem. The absorption spectrum of PeP compound I closely resembles that observed for the HRP compound I species. The m.c.d. data for PeP I clearly identifies that ring oxidation has occurred. One-electron reduction forms the PeP compound II species. The absorption and m.c.d. spectra recorded for PeP II exhibit the well-resolved spectral characteristics previously observed for both HRP compound II and catalase compound II. The spectral data of PeP with HRP and catalase are compared. The data clearly indicate that the m.c.d. spectral patterns of both plant peroxidases (PeP and HRP) are very similar and, therefore, the electronic structures of their resting states, and as well their primary and secondary compounds, must be similar. The m.c.d. data suggest that, while the compound I species of PeP and HRP belong to one electronic class, catalase compound I belongs to a different class. These data emphasize how the ground states of these two classes of oxidized haem, may be characterized as predominantly 2A2u (PeP I and HRP I) or 2A1u (catalase I). Peanut peroxidase is the second plant peroxidase for which the electronic structure of the compound I intermediate has been studied using the m.c.d. technique. The similarities with horseradish peroxidase allow us to suggest that plant peroxidases may operate by the same general mechanism, in spite of the low degree of sequence similarity between their polypeptide chains.
Animals↗
Chiral copper(I)-thiolate clusters in metallothionein and glutathione.
Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal-thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M7S20 structure are M4(Scys)11 (alpha domain) and M3(Scys)9 (beta domain). Up to 12 Cu(I) ions can displace the 7 Zn(2+) ions bound per molecule in Zn-MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu12S20 structure of Cu6(Scys)11 and Cu6(Scys)9 for the alpha and beta domains, respectively. The circular dichroism (CD) spectra recorded as Cu+ is added to Zn-MT to form Cu12-MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(i); because the Cu(I)-thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)-MT species. Model Cu(I)-thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)-thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)-thiolate clusters in the alpha and beta domains of Cu12-MT are proposed.
Animals↗
Hypobaric hypoxia impairs spatial memory in an elevation-dependent fashion.
The effects of various levels of hypobaric hypoxia, exposure to reduced atmospheric pressure, on spatial memory in rats were examined. Hypobaric hypoxia simulates high altitude conditions where substantial deficits in human cognitive performance occur. However, few studies have measured cognitive changes in animals during exposure to this type of hypoxia. Male Fischer 344 rats were tested in the learning set version of the Morris water maze, a test known to assess spatial memory. Rats were tested at 2 and 6 hours while exposed to a range of simulated altitudes: sea level, 5500 m, 5950 m, and 6400 m. Altitude exposures at 5950 or 6400 m decreased both reference and working memory performance, as demonstrated by latency, distance, and speed measures, in an elevation-dependent fashion. During sea level testing on the day following hypobaric exposure, decrements in reference memory were still observed on all dependent measures, but only speed was impaired on the working memory task. These results agree with human studies that demonstrate elevation-dependent impairments in spatial memory performance during exposure to hypobaric hypoxia. The deficits may be attributable to changes in hippocampal cholinergic function.
Altitude↗
Nimodipine prevents the in vivo decrease in hippocampal extracellular acetylcholine produced by hypobaric hypoxia.
Hypoxia decreases acetylcholine (ACh) synthesis and release in vitro, and ACh synthesis in vivo; however, its effect on extracellular concentration of ACh in vivo is not known. The calcium channel blocker nimodipine is a cerebrovascular dilator which also increases extracellular ACh in vivo. Therefore, it may provide protection from the effects of hypobaric hypoxia on the cholinergic system either via its effects on vascular function or by direct action on the nervous system. This study examined the effect of hypobaric hypoxia on extracellular ACh and choline levels, as measured by microdialysis, as well as the effects of nimodipine under hypoxia. Microdialysis guide cannulae were implanted into the hippocampal region of male Fischer rats so that probes would sample from the CA1 and DG regions. Animals were then exposed for eight hours to a simulated altitude of 5,500 m (18,000 ft) or tested at sea level for an equivalent duration. HPLC with electrochemical detection was used for analysis of the dialysates. At 5,500 m extracellular ACh levels in the placebo-treated group were significantly lower than the sea level group values. This decrement was reversed by nimodipine administered i.p. immediately preceding altitude ascent (10 mg/kg) and 250 min post-altitude ascent (10 mg/kg). These data suggest that nimodipine may provide protection from the detrimental effects of hypoxia on hippocampal cholinergic function.
Acetylcholine↗
Co-dependency of calcium and porphyrin for an integrated molecular structure of peanut peroxidase: a circular dichroism analysis.
The contribution of calcium to the structure of cationic peanut peroxidase was examined using ultraviolet/visible and circular dichroism spectroscopies under conditions in which the 2 moles of Ca2+ bound per mole of enzyme were removed. Cadmium and terbium ions were used as substitutes for calcium in the calcium depleted peroxidase and their influence on the protein structure was examined spectroscopically and compared to native and heme depleted enzymes. A role for the calcium ions in maintaining the active conformation of the peroxidase is proposed.
Arachis↗
Elevation of hippocampal extracellular acetylcholine levels by methoctramine.
Previous studies suggest that m2 muscarinic receptors serve as presynaptic autoreceptors. Blocking these receptors by selective antagonists may, therefore, lead to increased acetylcholine (ACh) release. This study assessed changes in extracellular ACh levels, via in vivo microdialysis, following administration of the m2 antagonist methoctramine. Drug or placebo (Ringer's solution) was perfused via a microdialysis probe into the CA1 hippocampal region of unrestrained, awake male Fischer rats. HPLC-EC was used for online analysis of the dialysates. Methoctramine significantly enhanced ACh release in a dose-dependent fashion as compared to placebo for the doses employed (0.25-16 microM). The present in vivo data corroborate studies that show increased ACh levels in vitro following application of m2 antagonists.
Acetylcholine↗
Retinal migraine, chorea, and retinal artery thrombosis in a patient with primary antiphospholipid antibody syndrome.
We report the case of a patient with the unusual combination of migraine, chorea, and retinal arterial thrombosis along with laboratory evidence of autoimmunity. In the absence of systemic lupus erythematosus, the clinical manifestations suggest the presence of the primary antiphospholipid antibody syndrome.
Adult↗