The role of agonist-independent conformational transformation (AICT) in IP₃ cluster behavior

Inositol 1,4,5-trisphosphate (IP(3)) receptor is a central unit in intracellular Ca(2+) signaling. Regulation of the IP? receptor by calcium is well characterized. High open probability values are reported for a single IP? receptor in nuclear patch clamp experiments. These experimental observations are in contrast with the lower open probability values of the lipid bilayer experiments. Most theoretical models do not account for high open probabilities of the receptor. But more recently, new models of the IP? receptor have been put forward which are constrained by single-channel nuclear patch clamp recordings, which generate the larger open probability with the aid of an additional agonist-independent conformational transformation (AICT)-'active' state. The main aim of this work is to constrain the AICT models with a wealth of experimental data characterizing calcium release from IP? receptor clusters. Our results suggest that consistency of cluster release between theory and experiments constrains the kinetics of the agonist-independent conformational transition rates (AICT) to values which lead to small open probabilities for the IP? receptor inconsistent with nuclear patch clamp experimental data.     

M?ssbauer studies on the metal-thiolate cluster formation in Fe(II)-metallothionein

The stepwise 57Fe(II)-thiolate cluster formation in rabbit liver metallothionein-2 (MT) has been followed at pH 8.5 using M?ssbauer spectroscopy. The zero-field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting delta EQ and isomer shift delta as found for reduced rubredoxin (Rdred) or the model compound [Fe(II)(SPh)4]2-, thus indicating an Fe(II)-tetrathiolate coordination. A similar conclusion was reached also in previous electronic absorption studies [M. Good and M. Vasák (1986) Biochemistry 25,8353--8356]. The M?ssbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin-Hamiltonian formalism resulting in M?ssbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2-. The identity of the M?ssbauer parameters of partially and fully metal-occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal-occupied 57Fe(II)4-MT form appear to be magnetically isolated, whereas in the fully metal-saturated 57Fe(II)7-MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existence of three isolated metal binding sites and a metal-thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187, 125-129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704-710], which revealed two metal-thiolate clusters containing three and four metal ions, respectively, and involving all 20 cysteine residues in metal binding, the appearance of M?ssbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7-MT is peculiar and deserves further studies. It is concluded, moreover, that the four-metal cluster is diamagnetic with the four Fe(II) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two-step process.     

Zur Ern?hrung und Nahrungswahl des Uhus(Bubo bubo)

Zusammenfassung 1. Von 1969 bis 1973 wurden an 52 Uhubrutplätzen der Fränkischen Alb Reste von 8766 Wirbeltieren gesammelt. Von 25 Horstplätzen liegen Aufsammlungen von über 100, meist über 250 Beutetieren vor.2. Die Vollständigkeit der Skelettelemente in Gewöllen ist unterschiedlich und hängt z. T. von der Größe der Beutetiere ab. Bei Vögeln ist der Iarsometatarsus am häufigsten vertreten und zur Auszählung der Individuen besonders geeignet.3. Der Stückzahl nach führten Igel, Feldmaus, Ratte und Rebhuhn die Beuteliste der Population an; Feldhase, Igel und Ratte machten bereits über 50% des gesamten Beutegewichtes aus. Insgesamt spiegelt die Beuteliste ein vielfältiges Angebot einer reich gegliederten und wenig intensiv genutzten Kulturlandschaft wider.4. In einzelnen Horstlisten (n=54) waren Rebhuhn, Waldohreule, Feldmaus, Igel und Wanderratte am regelmäßigsten vertreten.5. Beutetiere verschiedener Größe und Herkunft konnten in einzelnen Gewöllen nebeneinander nachgewiesen werden, so daß wahrscheinlich ist, daß die meisten Uhus auf einzelnen Jagdflügen verschiedene Biotope berühren und dabei nicht auf einen Beutetyp fixiert sind. Bestimmte biotopbedingte Kombinationen kommen häufiger vor. Feldmausjagd scheint im Gegensatz zur Erbeutung vonApodemus-Arten systematischer betrieben zu werden.6. Beutespektren einzelner Horstreviere zeigen eine sehr unterschiedliche Bevorzugung einzelner Beutetiere. Bereits innerhalb einer Population können die Extreme weit auseinander liegen, sind aber in der Regel durch Zwischenwerte miteinander verbunden. Ausgesprochene Spezialisierung konnte in der großen Population nicht beobachtet werden. Auch der Anteil der Vögel an der Beute ist kein Charakteristikum, dessen vergleichende Analyse zu besonderen Schlüssen über standortsbedingte Beutewahl oder gar konstante geographische Unterschiede führen würde.7. Jahreslisten einzelner Horstplätze unterscheiden sich ebenfalls nicht unerheblich wie auch die Anteile bevorzugter Beutetiere. Schwankungen von Jahr zu Jahr innerhalb eines Reviers scheinen jedoch geringer zu sein als Unterschiede in benachbarten Horstrevieren ähnlicher Biotopzusammensetzung. Insgesamt werden Beutetiere offener Biotope bevorzugt gejagt.8. Zu überregionalen Vergleichen sind nur umfassende Beutelisten brauchbar, da kleine Stichproben zu sehr mit Zufälligkeiten behaftet sind. In 19 Beutelisten europäischer Uhus spielen jedoch nur wenige Beutetiere eine entscheidende Rolle; im allgemeinen dominieren Säugetiere.9. Ähnlichkeiten in der Zusammensetzung der Nahrung können über größere Entfernungen relativ hoch, zwischen benachbarten Populationen jedoch sehr gering sein. Ökologische und geographische Einflüsse verzahnen sich in vielfältiger Weise. Dies ist bei überregionalen Vergleichen zu beachten.10. Eine Einteilung der Beutetiere nach ökologischen Gesichtspunkten (z. B. Trophie-Ebenen) zeigt, daß der Uhu in der Regel überwiegend von Herbivoren lebt, in Gebieten mit geringer pflanzlicher Produktion (z. B. Küste, Hochgebirge) auf Beutetiere anderer trophischer Positionen (z. B. Omnivore) oder Ökosysteme (z. B. Seevögel) ausweichen kann.11. Der Uhu kann je nach Situation als Generalist oder mehr oder minder als Spezialist der Beutewahl gelten. Freiwillige Umstellung auf hohes Angebot einer Art ist wahrscheinlich mit Erhöhung der Effizienz des Beuteerwerbs verbunden. Davon zu unterscheiden ist durch äußere Umstände erzwungene Stenophagie. Der Fähigkeit einer Einengung des Nahrungsangebotes bis zu einem gewissen Grad zu folgen, hat für eine Eule mit hohem Nahrungsbedarf und ausgeprägter Ortstreue hohen Überlebenswert. Die Einstellung auf wenige Beutetiere hoher Abundanz befähigt den Uhu auch zur Nutzung intensiv bewirtschafteten Kulturlandes.

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On the diet of the eagle owl (Bubo bubo)

Summary 1. In 52 Territories of the eagle owl in Northern Bavaria remains of 8766 vertebrates were collected from 1969 to 1973. From 25 nesting sites collections contain more than 100, mostly more than 250 prey animals.2. The completeness of scleton elements in pellets is different depending partly on the size of the prey animal. In birds the tarsometatarsus is the commonest bone in pellets and therefore represents the number of prey items rather well (table 1 and 2).3. Hedgehog, field vole, rat, and partridge were taken most often. The amount of biomass of hedgehog, hare, and rat was more than 50 percent of the whole prey. The diet of the population investigatet represents the fauna of a highly diverse and extensively used countryside (table 3).4. In the diet of single territories (n=54) partridge, long-eared owl, field vole, hedgehog and rat were represented most regularly (table 4).5. Within single pellets prey animals of different habitats and size could be found together. So it seems likely that individual eagle owls hunt in different habitats and do not prey on distinct types of prey animals. Certain combinations could be stated more often than others. It seems that field voles are hunted more systematically thanApodemus-species (fig. 1).6. Certain prey species were represented to a different amount in diets of different nesting sites. Within one population the percentages of one prey species can vary considerably from year to year and from territory to territory. Between extreme positions, however, many intermediate values could be found (figure 3 and 4). Extreme specialisation could not be found, even the percentages of birds in the diet vary widely and are no characteristical parameter for feeding habits of a given population (figure 5).7. Within one territory yearly diets vary remarkably as well as the percentages of favorite prey species. Yearly changes, however, seem to be smaller than differences between different territories in a similar habitat in the same year (table 7). Species of open habitat form the main diet.8. For comparisons between different populations only comprehensive lists are useful whereas small samples show to many errors. In the diet of 19 European eagle owl populations only a few prey species form the bulk (Table 8). As a rule mammals dominate.9. Between distant populations similarities of the diet could be found whereas the prey lists of neighbouring populations may be sometimes quite different. Ecological and geographical influences are integrated in many ways. This must be considered when comparing prey lists.10. Dividing the prey species according to trophic levels it becomes obvious that the eagle owl is preying mainly on herbivorous species. In areas with a low production of plants (i.e. coastal rocks, high mountains) the eagle owl is able to change over to animals of other trophic positions (i. e. omnivorous species) or other ecosystems (i. e. sea birds; figure 10 and 11).11. According to the ecological situation the eagle owl may be considered as a food generalist or as a food specialist. Spontaneous specialising in preying on a species very abundant most likely favours successful hunting. This must be separated from stenophagy forced by environmental circumstances. The ability of adapting to a smaller variety of prey species is necessary for a sedentary owl with high food consumption. The exploition of very abundant resources consisting of only a few species also enables the eagle owl to hunt in intensively used monotonous cultivated areas.

     

Copy number polymorphism in the α-globin gene cluster of European rabbit (Oryctolagus cuniculus)

Comparative genomic studies have revealed that mammals typically possess two or more tandemly duplicated copies of the α-globin (HBA) gene. The domestic rabbit represents an exception to this general rule, as this species was found to possess a single HBA gene. Previous electrophoretic surveys of HBA polymorphism in natural populations of the European rabbit (Oryctolagus cuniculus) revealed extensive geographic variation in the frequencies of three main electromorphs. The variation in frequency of two electromorphs is mainly partitioned between two distinct subspecies of European rabbit, and a third is restricted to the hybrid zone between the two rabbit subspecies in Iberia. Here we report the results of a survey of nucleotide polymorphism, which revealed HBA copy number polymorphism in Iberian populations of the European rabbit. By characterizing patterns of HBA polymorphism in populations from the native range of the European rabbit, we were able to identify the specific amino-acid substitutions that distinguish the previously characterized electromorphs. Within the hybrid zone, we observed the existence of a second HBA gene duplicate, named HBA2, that mostly represents a novel sequence haplotype, which occurs in higher frequency within the hybrid zone, and thus appears to have arisen in hybrids of the two distinct subspecies. Although this novel gene is also present in other wild Iberian populations, it is almost absent from French populations, which suggest a recent ancestry, associated with the establishment of the post-Pleistocene contact zone between the two European rabbit subspecies.     

Does 13C-or 15N-labeling affect Cu(I)-thiolate cluster arrangement in yeast copper-metallothionein?

It was attempted to examine whether or not isotope labeling may possibly affect an oligonuclear metal-thiolate cluster. Cu-metallothioneins are known to contain strongly distorted Cu-thiolate clusters and seemed appropriate for this study. Thus, yeast ¹³C-and ¹⁵N-Cu-metallothioneins were isolated from Saccharomyces cerevisiae cells grown in a minimal synthetic medium and some physicochemical parameters were compared with those of the unlabeled Cu-thionein. Surprisingly, the ¹³C- and ¹⁵N- labeled Cu₇-thioneins are distinctly different in their characteristic spectroscopic properties. The electronic absorption was blue-shifted while both luminescence emission and chiroptic features display a distinct red shift with markedly diminished intensities, respectively. Contrary to common knowledge that isotope labeling does not affect the molecular architecture of a protein the present results support such a phenomenon. Attributable to the fortunate happenstance that there is a strongly distorted structural situation in the oligonuclear Cu-thiolate cluster this isotope effect came to light.     

Ferromagnetic exchange coupling in the linear,chloride-bridged cluster (cyclam)CoII[(μ-Cl)UIV(Me2Pz)4]2

The trinuclear cluster (cyclam)Co[(μ-Cl)U(Me₂Pz)₄]₂ (cyclam = 1,4,8,11-tetraazacyclotetradecane, Me₂Pz^? = 3,5-dimethylpyrazolate) is synthesized through cleavage of the homoleptic dimer [U(Me₂Pz)₄]₂ by (cyclam)CoCl₂. A single crystal X-ray diffraction analysis reveals a linear chloride-bridged structure analogous to that previously reported for (cyclam)M[(μ-Cl)U(Me₂Pz)₄]₂ (M = Ni, Cu, Zn). The magnetic exchange coupling of the CoU₂ cluster was probed by analyzing the temperature dependence of its magnetic susceptibility. Comparison of χ_MT versus T between the CoU₂ species and the diamagnetic ZnU₂ cluster demonstrates the presence of ferromagnetic coupling between the Co^II and U^IV centers. We present methods for estimating upper and lower bounds for the exchange interaction energy in such systems and find that for CoU₂, the exchange constant, J, lies in the range 15–48 cm^?1. Application of these methods to the previously reported NiU₂ cluster suggests somewhat weaker ferromagnetic exchange, with J lying in the range 2.8–19 cm^?1. AC magnetic susceptibility experiments were not indicative of single-molecule magnet behavior for the CoU₂ cluster, although qualitative interpretation of the low-temperature magnetization data suggests the presence of significant zero-field splitting in the ground state.     

Gene evolution in the chicken β-globin cluster

We have determined the cDNA sequence of the chicken embryonic β-like ?-globin gene. Comparison with the sequences of the chicken ρ-globin and β-globin genes reveals the presence of two regions that are identical or nearly identical in ? and ρ. The first contains the 5′ untranslated sequence and exon 1, while the second region includes the second half of axon 2. Outside these regions ρ and ? are less homologous to each other than to the adult β-globin gene. The embryonic ρ and ? genes are located at opposite ends of the β-globin-gene cluster, not contiguously as are all other known pairs of simultaneously expressed globin genes. We suggest a role for gene conversion in the synchronization of expression of two highly diverged genes.     

Iron–sulfur cluster biosynthesis in photosynthetic organisms

Iron–sulfur (Fe/S) cluster containing proteins are widely distributed in nature and are involved in numerous processes including electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression. The knowledge about the biogenesis of Fe/S clusters, and the assembly and maturation of Fe/S cluster containing proteins is still limited, especially in photosynthetic organisms. In most organisms analyzed so far the biogenesis of Fe/S clusters involves more than one machinery. The additional compartment in photoautotrophic organisms, the plastids, presents an additional challenge for the regulation of Fe/S cluster biogenesis. The requirement for Fe/S proteins in multiple chloroplast processes argues that Fe/S cluster assembly is an essential part of plastid functionality. This review focuses on the interesting and unique aspects of Fe/S cluster biogenesis in photosynthetic organisms and compares them to what is known in other organisms.     

Iron–sulfur cluster scaffold (ISCU) gene is duplicated in salmonid fish and tissue and temperature dependent expressed in rainbow trout

The iron–sulfur cluster protein ISCU is a scaffold protein tasked with the building and mediation of iron–sulfur [Fe–S]-clusters. These are crucial for [Fe–S]-enzymes, which are involved in essential biological cell processes like metabolism or ion transport. Analysis of ISCU in rainbow trout (Oncorhynchus mykiss) and maraena whitefish (Coregonus maraena) revealed the existence of two gene variants in each of the two salmonids. This study presents the characterization of the duplicated ISCU cDNA sequences in both species as well as the comparative functional analysis of the genes in healthy and affected fish of two rainbow trout strains differing in trait robustness under regional aquaculture conditions. Coding sequences of trout ISCUA and ISCUB genes are spanning over five exons. Open reading frames (ORF) of trout (ISCUA: 495 bp, ISCUB: 498 bp) and whitefish (ISCUA and ISCUB: 495 bp) genes encode for evolutionary highly conserved proteins and share 72% sequence similarity with human ISCU.     

Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum : sequence, operon structure and mutational analysis of an exo gene cluster

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4′-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant ΔP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed.     

Comparison of theα-globin gene cluster structure in Perissodactyla

Summary To investigate molecular evolution in a mammalian order with a comprehensive fossil record, we have constructed-globin-like gene cluster maps for members of the order Perissodactyla. Although the arrangement of genes is the same in the five Equidae examined, the tapir and rhinoceros differ from each other and the horse in the position and number of their genes, but not in the arrangement of their and genes. In contrast to morphological work, a dendrogram derived from restriction site maps associates the tapir with the horse rather than with the rhinoceros; however, this phylogeny is not statistically significant. Among the Equidae,Equus caballus emerges as an outgroup, in agreement with data from other disciplines.     

Vegetation change in created emergent wetlands (1988–1996) in Connecticut (USA)

Changes in hydrology, water quality and vegetation were evaluated in four palustrine emergent wetland pairs, each including created and reference sites. Located along interstate highways, they were initially sampled in 1988 (Confer and Niering, 1992) and again in 1996. Overall, created sites showed significant decreases in open water and water depth between 1989 and 1996 compared to more stable conditions in reference sites. Total nitrogen was generally higher in created sites compared to reference sites, as was specific conductivity, with chloride levels exceeding 800 mg/L, apparently related to road salt. Emergent plant cover increased from 30 to 39% at three created sites, and decreased at a fourth, whereas reference sites remained relatively stable. Wetland species richness also increased from 31 to 39 species at created sites and 35 to 42 species at reference wetlands between the surveys. By 1996 there was an increase in invasive species, particularly Phragmites australis (common reed) and Lythrum salicaria (purple loosestrife). Phragmites increased from <1 to 15% at created sites, while Lythrum increased at one reference site from <1 to 16%. Typha latifolia (common cattail), dominant in the created wetlands in 1988, decreased from 16 to 5% while Typha angustifolia (narrow-leaved cattail) increased from 2 to 10%. At two created sites experiencing increased sedimentation, Phragmites is now dominant or co-dominant with Typha spp., whereas Carex stricta (tussock sedge) and T. latifolia continue to dominate at reference sites. At the one created, permanent flow-through-hydrology wetland, a three-fold decrease in T. latifolia and an eight-fold increase in Phragmites cover have occurred, the latter correlated with sedimentation from road culverts. Increases in alien or invasive species such as Phragmites and Lythrum can serve as indicators of wetland disturbance. Although these created wetlands provide the services of sediment retention, flood storage, and wildlife habitat, a greater range of wetland functions should be possible by constructing two-tiered systems that remove excess sediments and nutrients upstream of the wetland designed to compensate for wetland loss.     

IUBMB Symposia in 2000(英文)

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Evolution of the β-globin gene cluster in man and the primates

The arrangement of primate β-related globin genes has been determined by restriction endonuclease mapping of genomic DNA from species ranging from prosimians to man. The arrangement of the entire ?γγδβ-globin gene cluster in the gorilla and the yellow baboon is indistinguishable from that of man. Restriction site differences between these species are consistent with a surprisingly low overall rate of intergenic DNA sequence divergence of approximately 1% in 5 million years. A new world monkey (owl monkey) has a single γ-globin gene, suggesting that the ^Gγ-^Aγ-globin gene duplication in man is ancient, and occurred about 20 to 40 million years ago. The β-globin gene cluster in the brown lemur, a prosimian, is remarkably short (about 20,000 base-pairs) and contains single ?-, γ- and β-globin genes. The γ- and β-globin genes in this animal are separated by a curious gene containing the 3′ end of a β-globin gene preceded by sequences related to the 5′ end of the ?-globin gene.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.