Retrovirus particles with type C morphology were found in two T-cell lymphoblastoid cell lines, HUT 102 and CTCL-3, and in fresh peripheral blood lymphocytes obtained from a patient with a cutaneous T-cell lymphoma (mycosis fungoides). The cell lines continuously produce these viruses, which are collectively referred to as HTLV, strain CR(HTLV(CR)). Originally, the production of virus from HUT 102 cells required induction with 5-iodo-2'-deoxyuridine, but the cell line became a constitutive producer of virus at its 56th passage. Cell line CTCL-3 has been a constitutive producer of virus from its second passage in culture. Both mature and immature extracellular virus particles were seen in thin-section electron micrographs of fixed, pelleted cellular material; on occasion, typical type C budding virus particles were seen. No form of intracellular virus particle has been seen. Mature particles were 100-110 nm in diameter, consisted of an electron-dense core surrounded by an outer membrane separated by an electron-lucent region, banded at a density of 1.16 g/ml on a continuous 25-65% sucrose gradient, and contained 70S RNA and a DNA polymerase activity typical of viral reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase). Under certain conditions of assay, HTLV(CR) RT showed cation preference for Mg(2+) over Mn(2+), distinct from the characteristics of cellular DNA polymerases purified from human lymphocytes and the RT from most type C viruses. Antibodies to cellular DNA polymerase gamma and anti-bodies against RT purified from several animal retroviruses failed to detectably interact with HTLV(CR) RT under conditions that were positive for the respective homologous DNA polymerase, demonstrating a lack of close relationship of HTLV(CR) RT to cellular DNA polymerases gamma or RT of these viruses. Six major proteins, with sizes of approximately 10,000, 13,000, 19,000, 24,000, 42,000, and 52,000 daltons, were apparent when doubly banded, disrupted HTLV(CR) particles were chromatographed on a NaDodSO(4)/polyacrylamide gel. The number of these particle-associated proteins is consistent with the expected proteins of a retrovirus, but the sizes of some are distinct from those of most known retroviruses of the primate subgroups.

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular, ROS activation of AP-1 (activator protein) and NF-kappaB (nuclear factor kappa B) signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu, Zn-SOD, Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of carcinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappaB, AP-1 are also reviewed.

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.

Neuromotor prostheses (NMPs) aim to replace or restore lost motor functions in paralysed humans by routeing movement-related signals from the brain, around damaged parts of the nervous system, to external effectors. To translate preclinical results from intact animals to a clinically useful NMP, movement signals must persist in cortex after spinal cord injury and be engaged by movement intent when sensory inputs and limb movement are long absent. Furthermore, NMPs would require that intention-driven neuronal activity be converted into a control signal that enables useful tasks. Here we show initial results for a tetraplegic human (MN) using a pilot NMP. Neuronal ensemble activity recorded through a 96-microelectrode array implanted in primary motor cortex demonstrated that intended hand motion modulates cortical spiking patterns three years after spinal cord injury. Decoders were created, providing a 'neural cursor' with which MN opened simulated e-mail and operated devices such as a television, even while conversing. Furthermore, MN used neural control to open and close a prosthetic hand, and perform rudimentary actions with a multi-jointed robotic arm. These early results suggest that NMPs based upon intracortical neuronal ensemble spiking activity could provide a valuable new neurotechnology to restore independence for humans with paralysis.

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Photosynthesis uses light energy to drive the oxidation of water at an oxygen-evolving catalytic site within photosystem II (PSII). We report the structure of PSII of the cyanobacterium Thermosynechococcus elongatus at 3.5 angstrom resolution. We have assigned most of the amino acid residues of this 650-kilodalton dimeric multisubunit complex and refined the structure to reveal its molecular architecture. Consequently, we are able to describe details of the binding sites for cofactors and propose a structure of the oxygen-evolving center (OEC). The data strongly suggest that the OEC contains a cubane-like MnMn by a mono-micro-oxo bridge. The details of the surrounding coordination sphere of the metal cluster and the implications for a possible oxygen-evolving mechanism are discussed.

Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 Å. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 Å. From our electron density map, we located all of the metal atoms of the Mn(4)CaO(5) cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn(4)CaO(5) cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.

The Sod2 gene for Mn-superoxide dismutase (MnSOD), an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation, was inactivated by homologous recombination. Homozygous mutant mice die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis. Cytochemical analysis revealed a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide.

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.

Micropipette pressurization of giant bilayer vesicles was used to measure both elastic bending k(c) and area stretch K(A) moduli of fluid-phase phosphatidylcholine (PC) membranes. Twelve diacyl PCs were chosen: eight with two 18 carbon chains and degrees of unsaturation from one double bond (C18:1/0, C18:0/1) to six double bonds per lipid (diC18:3), two with short saturated carbon chains (diC13:0, diC14:0), and two with long unsaturated carbon chains (diC20:4, diC22:1). Bending moduli were derived from measurements of apparent expansion in vesicle surface area under very low tensions (0.001-0.5 mN/m), which is dominated by smoothing of thermal bending undulations. Area stretch moduli were obtained from measurements of vesicle surface expansion under high tensions (>0.5 mN/m), which involve an increase in area per molecule and a small-but important-contribution from smoothing of residual thermal undulations. The direct stretch moduli varied little (< +/-10%) with either chain unsaturation or length about a mean of 243 mN/m. On the other hand, the bending moduli of saturated/monounsaturated chain PCs increased progressively with chain length from 0.56 x 10(-19) J for diC13:0 to 1.2 x 10(-19) J for diC22:1. However, quite unexpectedly for longer chains, the bending moduli dropped precipitously to approximately 0.4 x 10(-19) J when two or more cis double bonds were present in a chain (C18:0/2, diC18:2, diC18:3, diC20:4). Given nearly constant area stretch moduli, the variations in bending rigidity with chain length and polyunsaturation implied significant variations in thickness. To test this hypothesis, peak-to-peak headgroup thicknesses h(pp) of bilayers were obtained from x-ray diffraction of multibilayer arrays at controlled relative humidities. For saturated/monounsaturated chain bilayers, the distances h(pp) increased smoothly from diC13:0 to diC22:1 as expected. Moreover, the distances and elastic properties correlated well with a polymer brush model of the bilayer that specifies that the elastic ratio (k(c)/K(A))(1/2) = (h(pp) - h(o))/24, where h(o) approximately 1 nm accounts for separation of the headgroup peaks from the deformable hydrocarbon region. However, the elastic ratios and thicknesses for diC18:2, diC18:3, and diC20:4 fell into a distinct group below the correlation, which showed that poly-cis unsaturated chain bilayers are thinner and more flexible than saturated/monounsaturated chain bilayers.

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian superoxide dismutases have been biochemically and molecularly characterized to date. SOD1, or CuZn-SOD (EC 1.15.1.1), was the first enzyme to be characterized and is a copper and zinc-containing homodimer that is found almost exclusively in intracellular cytoplasmic spaces. SOD2, or Mn-SOD (EC 1.15.1.1), exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces. SOD3, or EC-SOD (EC 1.15.1.1), is the most recently characterized SOD, exists as a copper and zinc-containing tetramer, and is synthesized containing a signal peptide that directs this enzyme exclusively to extracellular spaces. What role(s) these SODs play in both normal and disease states is only slowly beginning to be understood. A molecular understanding of each of these genes has proven useful toward the deciphering of their biological roles. For example, a variety of single amino acid mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis. Knocking out the SOD2 gene in mice results in a lethal cardiomyopathy. A single amino acid mutation in human SOD3 is associated with 10 to 30-fold increases in serum SOD3 levels. As more information is obtained, further insights will be gained.

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn(2+) or Mn(2+)) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn(2+)- and Mn(2+)-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn(2+)-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn(2+)-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn(2+)-Phos-tag SDS-PAGE).

The mucosal vascular addressin, MAdCAM-1, is an immunoglobulin superfamily adhesion molecule for lymphocytes that is expressed by mucosal venules and helps direct lymphocyte traffic into Peyer's patches (PP) and the intestinal lamina propria. We demonstrate that the lymphocyte integrin alpha 4 beta 7, also implicated in homing to PP, is a receptor for MAdCAM-1. Certain antibodies to alpha 4 and beta 7 integrin chains but not to the beta 2 integrin LFA-1 inhibit lymphocyte binding to purified MAdCAM-1 and to MAdCAM-1 transfectants. Lymph node lymphocytes, alpha 4 beta 7+ TK1 lymphoma cells, and a beta 7-transfected variant of an alpha 4+ B cell line, 38C13, bind constitutively to MAdCAM-1. Binding is enhanced by Mn(++)-induced integrin activation. The related integrin alpha 4 beta 1 supports efficient binding to VCAM-1 but not to MAdCAM-1, even after integrin activation, indicating that MAdCAM-1 is a preferential ligand for alpha 4 beta 7. Alpha 4 beta 7 can also bind VCAM-1, but this requires greater integrin activation than binding to MAdCAM-1. The findings imply a selective role for the interaction of alpha 4 beta 7 and MAdCAM-1 lymphocyte in homing to mucosal sites.

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.

Excessive reactive oxygen species Revised abstract, especially superoxide anion (O₂•-), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against (O₂•-), which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of (O₂•-), H2O2, which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SOD-dependent therapeutic strategies.

1. The electrical activity of Purkinje cells was studied in guinea-pig cerebellar slices in vitro. Intracellular recordings from Purkinje cell somata were obtained under direct vision, and antidromic, synaptic and direct electroresponsiveness was demonstrated. Synaptic potentials produced by the activation of the climbing fibre afferent could be reversed by direct membrane depolarization. 2. Input resistance of impaled neurones ranged from 10 to 19 M omega and demonstrated non-linearities in both hyperpolarizing and depolarizing directions. 3. Direct activation of a Purkinje cell indicated that repetitive firing of fast somatic spikes (s.s.) occurs, after a threshold, with a minimum spike frequency of about 30 spikes/sec, resembling the '2-class' response of crab nerve (Hodgkin, 1948). 4. As the amplitude of the stimulus was increased, a second form of electroresponsiveness characterized by depolarizing spike bursts (d.s.b.) was observed and was often accomppanied by momentary inactivation of the s.s. potentials. Upon application of tetrodotoxin (TTX) or removal of Na+ ions from the superfusion fluid, the s.s. potentials were abolished while the burst responses remained intact. However, Ca conductance blockers such as Co, Cd, Mn and D600, or the replacement of Ca by Mg, completely abolish d.s.b.s. 5. If Ca conductance was blocked, or Ca removed from the superfusion fluid without blockage of Na conductance, two types of Na-dependent electroresponsiveness were seen: (a) the s.s. potentials and (b) slow rising all-or-none responses which reached plateau at approximately -15 mV and could last for several seconds. These all-or-none Na-dependent plateau depolarizations outlasted the stimulus and were accompanied by a large increase in membrane conductance. Within certain limits the rate of rise and amplitude of the plateau were independent of stimulus strength. The latency, however, was shortened as stimulus amplitude was increased. These potentials were blocked by TTX or by Na-free solutions. 6. Substitution of extracellular Ca by Ba or intracellular injection of tetraethylammonium generated prolonged action potentials lasting for several seconds and showing a plateau more ositive than those obtained in norrmal circumstances by either non-inactivating Na or Ca currents. 7. Spontaneous firing of the Purkinje cell was characterized by burst-like activity consisting of both s.s. and d.s.b. responses. Addition of TTX to the bath left the basic spontaneous activity and its frequency unaltered, indicating tha Ca spiking and Ca-dependent K conductance changes are the main events underlying this oscillatory behaviour. 8...

Reactive O(2) species (ROS) are produced in both unstressed and stressed cells. Plants have well-developed defence systems against ROS, involving both limiting the formation of ROS as well as instituting its removal. Under unstressed conditions, the formation and removal of O(2) are in balance. However, the defence system, when presented with increased ROS formation under stress conditions, can be overwhelmed. Within a cell, the superoxide dismutases (SODs) constitute the first line of defence against ROS. Specialization of function among the SODs may be due to a combination of the influence of subcellular location of the enzyme and upstream sequences in the genomic sequence. The commonality of elements in the upstream sequences of Fe, Mn and Cu/Zn SODs suggests a relatively recent origin for those regulatory regions. The differences in the upstream regions of the three FeSOD genes suggest differing regulatory control which is borne out in the research literature. The finding that the upstream sequences of Mn and peroxisomal Cu/Zn SODs have three common elements suggests a common regulatory pathway. The tools are available to dissect further the molecular basis for antioxidant defence responses in plant cells. SODs are clearly among the most important of those defences, when coupled with the necessary downstream events for full detoxification of ROS.

A dissimilatory Fe(III)- and Mn(IV)-reducing microorganism was isolated from freshwater sediments of the Potomac River, Maryland. The isolate, designated GS-15, grew in defined anaerobic medium with acetate as the sole electron donor and Fe(III), Mn(IV), or nitrate as the sole electron acceptor. GS-15 oxidized acetate to carbon dioxide with the concomitant reduction of amorphic Fe(III) oxide to magnetite (Fe(3)O(4)). When Fe(III) citrate replaced amorphic Fe(III) oxide as the electron acceptor, GS-15 grew faster and reduced all of the added Fe(III) to Fe(II). GS-15 reduced a natural amorphic Fe(III) oxide but did not significantly reduce highly crystalline Fe(III) forms. Fe(III) was reduced optimally at pH 6.7 to 7 and at 30 to 35 degrees C. Ethanol, butyrate, and propionate could also serve as electron donors for Fe(III) reduction. A variety of other organic compounds and hydrogen could not. MnO(2) was completely reduced to Mn(II), which precipitated as rhodochrosite (MnCO(3)). Nitrate was reduced to ammonia. Oxygen could not serve as an electron acceptor, and it inhibited growth with the other electron acceptors. This is the first demonstration that microorganisms can completely oxidize organic compounds with Fe(III) or Mn(IV) as the sole electron acceptor and that oxidation of organic matter coupled to dissimilatory Fe(III) or Mn(IV) reduction can yield energy for microbial growth. GS-15 provides a model for how enzymatically catalyzed reactions can be quantitatively significant mechanisms for the reduction of iron and manganese in anaerobic environments.

1. Intradendritic recordings from Purkinje cells in vitro indicate that white matter stimulation produces large synaptic responses by the activation of the climbing fibre afferent, but antidromic potentials do not actively invade the dendritic tree. 2. Climbing fibre responses may be reversed in a manner similar to that observed at the somatic level. However, the reversal does not show the biphasicity often seen at somatic level. 3. Input resistance of these dendrites was found to range from 15 to 30 M omega. The non-linear properties seen at the somatic level for depolarizing currents are also encountered here. However, there seems to be less anomalous rectification. 4. Detailed analysis of repetitive firing of Purkinje cells elicited by outward DC current shows that, as in the case of the antidromic invasion, the fast somatic potentials (s.s.) do not invade the dendrite actively. However, the dendritic spike bursts (d.s.b.s) interposed between the s.s. potentials are most prominent at dendritic level. 5. Two types of voltage-dependent Ca responses were observed. At low stimulus level a plateau-like depolarization is accompanied by a prominent conductance change; further depolarization produces large dendritic action potentials. These two classes of response are TTX-resistant but are blocked by Cd, Co, Mn or D600, or by the removal of extracellular Ca. 6. Following blockage of the Ca conductance, plateau potentials produced by a non-inactivating Na conductance are observed mainly near the soma indicating that this voltage-dependent conductance is probably associated with the somatic membrane. 7. Spontaneous firing in Purkinje cell dendrites is very similar to that observed at the soma. However, the amplitude of these bursts is larger at dendritic level. It is further concluded that these TTX-insensitive spikes are generated at multiple sites along the dendritic tree. 8. Six ionic conductances seem to be involved in Purkinje cell electroresponsiveness: (a) an inactivating and (b) a non-inactivating Na conductance at or near the soma, (c) a spike- and (d) a plateau-generating Ca conductance, and (e) voltage-dependent and (f) Ca-dependent K currents. 9. The possible role of these conductances in Purkinje cell integration is discussed.

Evidence is given for a high density of negative surface charge near the sodium channel of myelinated nerve fibres. The voltage dependence of peak sodium permeability is measured in a voltage clamp. The object is to measure voltage shifts in sodium activation as the following external variables are varied: divalent cation concentration and type, monovalent concentration, and pH. With equimolar substitution of divalent ions the order of effectiveness for giving a positive shift is: Ba equals Sr less than Mg less than Ca less than Co approximately equal to Mn less than Ni less than Zn. A tenfold increase of concentration of any of these ions gives a shift of +20 to +25 mV. At low pH, the shift with a tenfold increase in Ca-2+ is much less than at normal pH, and conversely for high pH. Soulutions with no added divalent ions give a shift of minus 18 mV relative to 2 mM Ca-2+. Removal of 7/8 of the cations from the calcium-free solution gives a further shift of minue 35 mV. All shifts are explained quantitatively by assuming that changes in an external surface potential set up by fixed charges near the sodium channel produce the shifts. The model involves a diffuse double layer of counterions at the nerve surface and some binding of H+ions and divalent ions to the fixed charges. Three types of surface groups are postulated: (1) an acid pKa equals 2.88 charge density minus 0.9 nm- minus 2; (i) an acid pKa equals 4.58, charge density minus 0.58 nm- minus 2; (3) a base pKa equals 6.28, charge density +0.33 nm- minus 2. The two acid groups also bind Ca-2+ ions with a dissociation constant K equals 28 M. Reasonable agreement can also be obtained with a lower net surface charge density and stronger binding of divalent ions and H+ ions.

Photosystem II (PSII) is a large homodimeric protein-cofactor complex located in the photosynthetic thylakoid membrane that acts as light-driven water:plastoquinone oxidoreductase. The crystal structure of PSII from Thermosynechococcus elongatus at 2.9-A resolution allowed the unambiguous assignment of all 20 protein subunits and complete modeling of all 35 chlorophyll a molecules and 12 carotenoid molecules, 25 integral lipids and 1 chloride ion per monomer. The presence of a third plastoquinone Q(C) and a second plastoquinone-transfer channel, which were not observed before, suggests mechanisms for plastoquinol-plastoquinone exchange, and we calculated other possible water or dioxygen and proton channels. Putative oxygen positions obtained from a Xenon derivative indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in proton-transfer reactions because it is bound through a putative water molecule to the Mn(4)Ca cluster at a distance of 6.5 A and is close to two possible proton channels.

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.

Rat liver was homogenized in isotonic buffer, fractionated by differential centrifugation, and then subfractionated by equilibrium sedimentation in Nycodenz gradients. Fractions were assayed for both Cu,Zn-superoxide dismutase (SOD) and Mn-SOD by exploiting the cyanide sensitivity of the former activity and by the use of specific antibodies. As expected, the cytosol and lysosomal fractions contained Cu,Zn-SOD; while the mitochondrial matrix contained Mn-SOD. In mitochondria, Cu,Zn-SOD was found in the intermembrane space and Mn-SOD in the matrix and also on the inner membrane. The Mn-SOD associated with the inner membrane was solubilized by 0.5 m NaCl. Surprisingly the intracellular membrane fraction (microsomes) contained bound Cu,Zn-SOD that could be solubilized with a detergent, and to lesser degree with 0.5 m NaCl. Both the cytosolic and mitochondrial Cu,Zn-SODs were isolated and compared. They have identical molecular mass, cyanide sensitivity, SDS sensitivity, heat stability, and chloroform + ethanol stability. Tissue from Cu,Zn-SOD knockout mice was entirely devoid of Cu,Zn-SOD; indicating that the cytosolic and the intermembrane space Cu,Zn-SODs are coded for by the same gene. The significance of this distribution of the SODs is discussed.