Intact Cluster Research Articles

A Methodological Review of Non-Therapeutic Intervention Trials Employing Cluster Randomization, 1979–1989

A methodological review is presented of 16 non-therapeutic intervention trials published over the last decade which have randomized intact clusters rather than individuals in treatment groups. Each of the trials was surveyed as to the information supplied on six methodological criteria. Although there is increasing recognition of the methodological issues associated with cluster randomization, many investigators are still not aware of the impact of this design on sample size requirements and analysis considerations. Investigators are urged to publish the cluster-specific event rates observed in their trials as a guide for the planning of future studies.

Dynamics of boron cluster ion reactions with deuterium: Adduct formation and decay

Product cross sections for reaction of size-selected boron cluster ions with deuterium are reported for collision energies ranging from 0.05 to 10 eV. The major reactions are those in which one or two deuterium atoms add to the intact cluster. Other, higher energy channels involve fragmentation of the clusters, with and without addition of deuterium atoms. Cluster size dependent thresholds are observed for many reactions, and we have used these to estimate the cluster–D atom binding energy as a function of cluster size. Metastable adducts of the form BnD+2 are observed for reagent clusters as small as the tetramer. For small clusters, adducts form without activation energy, but starting at B+8 activation barriers are observed. Unimolecular decay rates of the adducts have been estimated, and the binding energy of D2 to various size cluster ions was extracted using RRKM modeling. Comparison is made with related results for boron surfaces and for aluminum cluster ions.

Evidence That the Iron-Sulfur Cluster of Bacillus subtilis Glutamine Phosphoribosylpyrophosphate Amidotransferase Determines Stability of the Enzyme to Degradation in Vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.

Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1.

The kps gene cluster of Escherichia coli K1 encodes functions for sialic acid synthesis, activation, polymerization, and possibly translocation of polymer to the cell surface. The size and complexity of this membrane polysaccharide biosynthetic cluster have hindered genetic mapping and functional descriptions of the kps genes. To begin a detailed investigation of the polysialic acid synthetic mechanism, acapsular mutants were characterized to determine their probable defects in polymer synthesis. The mutants were tested for complementation with kps fragments subcloned from two separately isolated, functionally intact kps gene clusters. Complementation was assayed by immunological and biochemical methods and by sensitivity to the K1-specific bacteriophage K1F. The kps cluster consisted of a central 5.8-kilobase region that contained at least two genes coding for sialic acid synthetic enzymes, a gene encoding the sialic acid-activating enzyme, and a gene encoding the sialic acid polymerase. This biosynthetic region is flanked on one side by an approximately 2.8-kilobase region that contains a potential regulatory locus and at least one structural gene for a polypeptide that appears to function in polysialic acid assembly. Flanking the biosynthetic region on the opposite side is a 6- to 8.4-kilobase region that codes for at least three proteins which may also function in polymer assembly and possibly in translocating polymer to the outer cell surface. Results of transduction crosses supported these conclusions and indicated that some of the kps genes flanking the central biosynthetic region may not function directly in transporting polymer to the cell surface. The results also demonstrate that the map position and probable function of most of the kps cluster genes have been identified.

The Camp Pearl Wheat Site 41KR23 A Program for Data Recovery

Site 41KR243, the Camp Pearl Wheat Site, is located along FM 783 and the south bank of Town Creek in Kerr County, central Texas (Fig. 1). The site was identified and recommended for testing by the State Department of Highways and Public Transportation (SDHPT) in response to plans for realigning a hazardous curve on FM 783. Test excavations were performed in June 1988 and consisted of three 2-meter square hand-excavated units within the proposed 120 foot wide right-of-way. Although testing was limited because a portion of the site is used as a summer youth camp, Camp Pearl Wheat, sufficient data was recovered to suggest that the site contains intact features at depths of 40 to 50 cm. A hearth eroding from an existing roadcut was recorded and an another intact burned rock cluster was found at a depth of 50 cm in Test Unit 1. The single diagnostic artifact from the testing phase, a Martindale dart point, closely resembled a dart point of the same style that was surface collected earlier when the site was originally identified. Testing indicated that site stratigraphy consisted of approximately 30 cm of dark gray silty clay intermixed with infrequent occurrences of subrounded limestone pebbles, overlying a dark brown silty loam. The 2-meter square test units were excavated in 10-cm levels that indicated the upper soil zone contained a very small amount of lithic debris. No burned rocks, bone, mussel shell, or stone tools were found. The absolute frequency of lithic debris per unit from this upper zone ranged from 1 to 25 and suggested an ephemeral temporary cultural presence. The lower dark brown silty clay level, however, the level which contained the exposed hearth and the Martindale dart point, had a higher frequency of lithic debris per level than the upper zone. Burned or fire-fractured rock was noted in all units within the deeper zone but no bone, mussel shell, or charcoal was observed. Although testing indicated that the prehistoric occupation was most intensive in the western half of the proposed right-of-way, the site did extend eastward across the right-of-way. Approximately 1000 square meters of relatively undisturbed site surface area lies within the project limits. Based on limited testing and intensive surface survey, an estimated 60% of the site is contained in the roadway right-of-way. Test Unit 3, the southernmost unit dug, was located either at or very near the southeastern margin of the site (Fig. 2). The original site dimensions, prior to disturbance by the first construction of FM 783, was estimated at approximately 70 x 40 meters.

Trout Sertoli and Leydig cells: isolation, separation, and culture.

Trout testes at various stages of maturation were dissociated by perfusion at 12 degrees C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two-step separation method: 1) the testis cell suspension was separated by sedimentation at unit gravity into "isolated cell" and "cell cluster" populations; 2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes). These various cell populations were cultured in modified Leibovitz L15 medium for 10-15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3 beta-HSD positive and produced progesterone and 17 alpha, 20 beta-OH progesterone for at least 11 days. This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.

Metal clusters and cluster metals

Abstract The desirable aim to create uniform naked metal clusters still fails. On the other hand, there exist many ligand stabilized clusters. If there was any possibility of eliminating the ligand sphere, a wide field of study, naked clusters of the same size, could be opened. Naked clusters with a magic number of metal atoms (full shell clusters) should have a special stability because of their complete outer geometry. Two-shell clusters of the type M55L12Clx (M = Au, Rh, L = PPh3, x = 6; M Rh, Ru, L = P(t-BU)3, x = 20, M = Pt, L = As(t-Bu)3, x = 20; M = Co, L = PMe3, x = 20) seem to be suited for studying those problems. As can be seen from HRTEM images and from other investigations, they have a cubic close packed structure (ccp) with the outer geometry of a cuboctahedron. The 12 vertices are coordinated by the 12 ligands L, and the chlorine atoms partially fill the gaps between the phosphine or arsine molecules. The nature of the figands makes the cluster molecules soluble in organic solvents. The transformation into water soluble compounds succeeds if PPh3 is exchanged by the hydrophilic derivative Ph2PC6H4SO3Na·2H2O. So, the cluster Au55(Ph2PC6H4SO3Na·2H2O)12Cl6 can be obtained as a very stable (in contrast to Au55(PPh3)12Cl16) water soluble compound which is completely dissociated in aqueous solution. Using a 100 kV electron beam, small crystalline areas of probably intact cluster molecules can be imaged by TEM. The giant cluster Pd56l(Phen)360190–200 (phen = phenanthroline) is obtained by reduction of palladium acetate with H2 in the presence of appropriate amounts of phen with subsequent oxidation by oxygen. The generation of naked clusters indeed succeeds if the M55L12Clx species are degraded in solution by 20 V d.c., using Pt electrodes. The ligand sphere together with the outer shell of metal atoms is eliminated, generating the inner naked M13 nuclei. These full-shell particles are metastable and build up novel structures. The structural principle can be described as an aggregation of 13 M13 particles, combined in a kind of close packed arrangement. The (M13)13 superclusters again act as building blocks and form particles [(M13)13]n. This construction principle is realized for all metals in this investigation, i.e. Au, Rh, Ru, Pt and Co.

Electron-transfer catalysis of ligand substitution in triiron clusters. The role of the bridging ligand in anion radical intermediates

The polynuclear cluster Fe/sub 3/(CO)/sub 9/(..mu../sub 3/-PPh)/sub 2/ (I) undergoes rapid ligand substitution by electron-transfer catalysis (ETC) under conditions in which the thermal process is nonexistent. X-ray crystallography and /sup 31/P NMR spectroscopy establish the stepwise substitution of the CO ligands by trimethyl phosphite to take place selectively at three separate iron centers. The high selectivity to the mono-substitution product II is achieved by tuning the reduction potential specifically to generate catalytic amounts of the anion radical Fe/sub 3/(CO)/sub 9/(PPh)/sub 2//sup -/ (I/sup -/) in either acetonitrile or tetrahydrofuran. Transient ESR spectroscopy of I/sup -/ and three related paramagnetic intermediates establish the sequential transformation of anion radicals as they evolve in the ETC mechanism. The rate-limiting rearrangement of I/sup -/ by the slippage of a phosphinidene cap from ..mu../sub 3/ ..-->.. ..mu../sub 2/ coordination underscores the key role of the bridging ligand in the substitution process. The importance of this critical transformation related to the formation of a 17-electron, coordinatively unsaturated iron center in the otherwise intact cluster. As such, it emphasizes the key role that the bridging ligand can play in cluster activation.

Ion-induced desorption by high-energy (600 keV) hydrogen clusters.

Desorption has been induced on CsI by hydrogen clusters of mass ranging from 5 to 23. A cluster effect has been observed in comparison of the ${\mathrm{Cs}}^{+}$ desorption yields as obtained from intact and preexploded clusters. The variation of the yield with the sum of the electronic stopping powers of the cluster components (additive stopping) shows a fourth-power dependence. The contribution of collective effects in the interaction may play a crucial role in the nonlinearity of the process.

Peptidergic neurons of Aplysia lose their response to cyclic adenosine 3':5'-monophosphate during a prolonged refractory period

Although the peptidergic bag cell neurons of Aplysia are ordinarily silent, they respond to brief electrical stimulation by producing an afterdischarge of about 30 min duration. This afterdischarge is followed by a refractory period lasting many hours during which electrical stimulation either fails to initiate afterdischarges or produces discharges of much shorter duration. Previous work has demonstrated that cyclic AMP plays a role in the genesis of afterdischarge, both in intact bag cell clusters and in isolated cultured bag cells. We have now examined the hypothesis that in the refractory period either the synthesis of cyclic AMP or the response to cyclic AMP is attenuated. Direct measurements of cyclic AMP showed that cyclic AMP levels in the bag cell neurons are elevated to a similar extent after stimulation in refractory and nonrefractory clusters of neurons. We have found, however, that the response to cyclic AMP is altered during the refractory period. The electrophysiological responses of bag cell neurons were first examined in intact clusters of cells within the abdominal ganglion. Cyclic AMP levels were elevated using the adenylate cyclase activator, forskolin, in the presence of theophylline (FT). The duration of a first bag cell afterdischarge could be greatly increased if FT was added before stimulation. The duration of a stimulated second bag cell afterdischarge could also be significantly increased if FT was added within a brief period following the end of the first afterdischarge. Furthermore, at these times the addition of FT often resulted in the onset of spontaneous afterdischarges.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotope labelling studies on the ruthenium-catalysed hydroformylation of ethylene: indirect evidence for catalysis at intact clusters

Isotope labelling studies suggest that the hydroformylation of ethylene catalysed by the cluster anion [Ru3H(CO)11]– proceeds through the intermediacy of intact Ru3 clusters.

Calcium entry causes a prolonged refractory period in peptidergic neurons of Aplysia

A brief train of electrical stimuli to the pleuroabdominal connective of Aplysia produces a cumulative depolarization in the peptidergic bag cell neurons within the abdominal ganglion. This response is followed by an afterdischarge which lasts for about 30 min, and then by a prolonged refractory period lasting for several hours. During the refractory period the cumulative depolarization in response to stimulation is attenuated, and stimulation either fails to initiate afterdischarges or produces discharges of much shorter duration. We have used the cationophore X537A to test the hypothesis that the prolonged refractory period is caused by calcium entry into the bag cell neurons during the afterdischarge. Exposure of intact bag cell clusters to X537A at concentrations from 1 to 10 microM for a period of 20 min in calcium-containing media produced no change in their resting potentials or in their ability to generate action potentials, but induced a state resembling natural refractoriness in response to subsequent stimulation. Both natural refractoriness and that induced by X537A could be overcome by extracellular tetraethylammonium ions (90 mM). Dose response data showed that concentrations of ionophore of 2.5 to 5.0 microM produce an attenuation of afterdischarge that is similar to that following a stimulated afterdischarge. These concentrations of X537A also produced an enhancement of 3H-labeled peptide release from these cells that is comparable to that observed on stimulation of an afterdischarge. Moreover, the time course of recovery from exposure to 5.0 microM X537A parallels that of natural refractoriness, recovery being essentially complete about 20 hr after X537A exposure or stimulation. The ionophore did not affect the mean duration of afterdischarge when applied in calcium-deficient media. The electrical effects of X537A were investigated using isolated bag cell neurons in cell culture. After treatment with the adenylate cyclase activator, forskolin, and theophylline, such isolated cells show many of the electrical changes occurring in intact bag cell clusters at the onset of afterdischarge, including the enhancement of action potentials, as well as increased input resistance and the emergence of oscillations in membrane potential. None of these parameters was significantly affected by concentrations of ionophore that induce refractoriness. In response to repetitive intracellular stimulation, however, some forskolin-treated cells undergo a cumulative depolarization which is similar to that seen at the onset of afterdischarge in intact clusters. This cumulative depolarization was found to be attenuated or abolished by 5.0 microM X537A.(ABSTRACT TRUNCATED AT 400 WORDS)

Support and cluster effects on synthesis gas conversion with supported ruthenium clusters

[Ru3(CO)12] and [H4Ru4(CO)12] supported on Al2O3 and MgO exhibited differences in catalytic behavior which can be attributed to the surface organometallic chemistry of the clusters. When supported on Al2O3, [Ru3(CO)12] decomposed to Ru metal on reaction with synthesis gas, and exhibited catalytic behavior that is similar to Ru/Al2O3 prepared by classical methods. On MgO, [Ru3(CO)12] resulted in significant CH3OH production from synthesis gas. After catalysis, the anionic cluster [Ru6C(CO)16]2− was identified on the catalyst, which results from the reaction of [HR3(CO)11]− and [Ru6(CO)18]2−, originally present on the support, with CO + H2. [H4Ru4-(CO)12] supported on Al2O3 and MgO also resulted in significant oxygenate yields from CO + H2. [H3Ru4(CO)12]− was originally present on the surface and reacts with synthesis gas to give [Ru6C(CO)16]2−. The oxygenate selectivity is suggested to be a result of the presence of intact metal clusters on the surface.

The role of iron in the activation-inactivation of aconitase.

Methods are described for the convenient preparation of aconitase from beef heart mitochondria in its inactive [3Fe-4S] form and largely in its active [4Fe-4S] form. Inactive aconitase can be activated anaerobically by various reducing agents without addition of iron. Under these conditions, maximally 70-80% of the activity attainable in the presence of added iron can be reached. It is concluded that during activation without added iron, [4Fe-4S] clusters are built from [3Fe-4S] clusters at the expense of a fraction of the 3Fe clusters present. This explains the approximately 75% maximal activation observed and concomitant loss of approximately 25% of total clusters as quantitated by EPR. Time course plots of aconitase activation appear to be second order but are not amenable to simple kinetic analysis because of the requirements of both reduction and Fe2+ for activation. Activation of aconitase with 59Fe leads to rapid (minutes) incorporation of 1 iron atom/cluster, which on subsequent inactivation is readily lost again. With longer incubation times (hour), 59Fe is found in more than a single site/cluster. It is concluded that, in analogy to cluster loss during activation in absence of added iron, the appearance of 59Fe in more than one cluster site can be due to complete breakdown and rebuilding of clusters. However, exchange into intact clusters cannot be ruled out. Ferric iron can be bound nonspecifically to active and inactive aconitase but can be readily removed by chelating agents. Sulfide is not required for activation of aconitase in keeping with the proposal that inactive aconitase, as isolated, contains a [3Fe-4S] cluster. It is demonstrated that oxidation initiates the inactivation of aconitase with concomitant release of iron and formation of 3Fe clusters as determined by EPR.

Time-resolved laser desorption mass spectrometry. I. Desorption of preformed ions

A laser desorption time-of-flight (LD-TOF) mass spectrometer has been used to study the time-resolved desorption of nonvolatile organic salts following a laser pulse. An electron beam, pulsed prior to the pulse which draws the ions from the source, can be used to examine the neutral species desorbed by the laser. Experiments on tetraalkylammonium halides indicate desorption rates for neutral decomposition products vastly different from those for intact ions and neutral clusters, so that the method can be used to resolve competing thermal processes.

Regulated expression of the human beta-globin gene family in murine erythroleukaemia cells.

Chemically induced differentiation of cultured murine erythroleukaemia (MEL) cells results in a several hundred-fold increase in transcription of the adult mouse globin genes and thus serves as a model for gene activation during erythropoiesis. One approach to study gene regulation in this system has been to analyse the expression of foreign globin genes introduced into MEL cells. By introducing cosmid DNA containing the human adult(beta), fetal(gamma) and embryonic(epsilon)-globin genes, we have shown here that expression of the beta, but not the gamma or epsilon genes, is regulated during MEL differentiation. Regulated expression of the human beta-globin gene was observed when it was introduced either as part of the intact globin gene cluster or as an individual gene with 1.5 kilobases (kb) of 5' flanking DNA. Transcription from a herpes simplex virus (HSV) promoter adjacent to the thymidine kinase (tk) gene is also inducible in MEL cells.

The honey bee cluster as a homeothermic superorganism

1. 1. In winter, oxygen consumption of honey bee ( Apis mellifera L.) clusters resembles that of birds and mammals. 2. 2. Intact clusters of 10–20,000 bees increase metabolism when exposed to cold environmental temperatures. Below 10°C, metabolic rate (W kg −1 ) increases as a function of decreasing ambient temperature following the relation MR = 7.96 — 0.24 T (Fig. 1). 3. 3. Insulative properties of the cluster are estimated and discussed. 4. 4. At moderate ambient temperatures (10–14°C), the cluster “breaks” resulting in a massive increase in total surface area for heat exchange and concomitant large increase in metabolism.

Photoinitiated Hydrosilations in Presence of Tetrahedral Heterometallic Clusters: Catalysis by Intact Clusters

Photoinitiated Hydrosilations in Presence of Tetrahedral Heterometallic Clusters: Catalysis by Intact Clusters

On the role of ubiquinone in the respiratory chain

(1) The V 1 (substrate-Q oxidoreductase activity) and V 2 (QH 2 oxidase activity) for the oxidation of substrates by submitochondrial particles have been measured by using heptylhydroxyquinoline N-oxide (HQNO) as inhibitor of V 2. (2) Partial destruction of the Rieske Fe-S cluster by treatment with BAL (2,3-dimercaptopropanol)+ O 2 has the same effect on the QH 2 oxidase activity as partial saturation of the antimycin-binding site with HQNO. (3) The extent of the rapid reduction of cytochrome b in the presence of excess antimycin is proportional to the percentage of intact Rieske Fe-S cluster. (4) The measured rate of oxidation of endogenous ubiquinol ( V 2) by submitochondrial particles is dependent on the substrate used to reduce ubiquinone, especially at low levels of ubiquinone. (5) Pool-function kinetics in the oxidation of substrate, found both in the presence and absence of free ubiquinone, are due both to the pool of free ubiquinone and to direct collision between Q-loaded Q-reducing and -oxidizing enzymes. At infinite Q content only the former mechanism is operative; at low Q content only the latter. (6) Duroquinone can be reduced directly by NADH dehydrogenase without mediation of ubiquinone, but duroquinol cannot be oxidized in the absence of ubiquinone. On the other hand, the reduction of cytochrome b by duroquinol does not require the presence of ubiquinone. (7) It is suggested that the need for ubiquinone for the oxidation of duroquinol is due to the requirement of ubisemiquinone for the oxidation of cytochrome b, duroquinol not being able to form a stabilized semiquinone.

Metabolic energy of intact honey bee colonies

1. 1. In late winter, oxygen consumption of honey bee ( Apis mellifera L.) clusters showed marked 24-hr periodicity, even when held under constant temperature conditions. 2. 2. Minimal rates of metabolism (as low as 3.4 w kg −1) were usually reached at night ( ca. 0500 hr), and maximum rates (as high as 33.5 w kg −1) in midday ( ca. 1400 hr). 3. 3. Colonies with brood showed less excursion in daily metabolic rate, by maintaining higher night-time levels. 4. 4. There is a pronounced decrease in metabolic rate for the intact cluster of 9480–23,394 bees from the rates reported for individuals or small groups of bees.