In VivoP NMR Studies of Corn Root Tissue and Its Uptake of Toxic Metals

Excised corn root tissue has been evaluated for its viability, integrity of compartmentation, intracellular pH gradients, total mobile phosphorus content and nucleotide concentrations under different levels of acidity, and mineral stresses using in vivo³¹P nuclear magnetic resonance spectroscopy at 21 to 23°C. Perfusion with Al³⁺ ion at low pH (4.0) for 20 hours caused the overall concentration of nucleotides in the cytoplasm to decrease significantly relative to the control. Respiratory activity as measured by O₂ uptake decreased by a comparable amount over this time period. The addition of glucose to the Al-containing perfusate negated the inhibitory effects on the respiratory system. Treatment of the tissue with paramagnetic manganese ion while perfusing in the presence of O₂ allowed for the observation of the sequence of events leading to the irreversible trapping of Mn²⁺ in the vacuole. Pretreatment of the roots with Mg²⁺ prevented Mn²⁺ migration to the vacuole over the time period of this experiment. Hypoxia prevented all but a limited uptake of Mn²⁺ into the cytoplasm of the root tips. No evidence of Mn²⁺ complexation of either cytoplasmic or vacuole Pi suggests that the energy derived from O₂ consuming processes is necessary for the facilitated movement of this divalent cation.     

Structural determinants of spermidine-DNA interactions

Twelve different analogs of spermidine (SPD) and SPD itself were compared for their ability to modulate two conformational transitions of DNA; the B-to-Z conformational transition of poly(dG-me⁵dC) and the thermal melting transition of calf thymus DNA. The analogs consisted of five N-ethyl-SPD derivatives [N¹-ethyl-SPD, N⁴-ethyl-SPD, N⁸-ethyl-SPD, N¹,N⁸-bis(ethyl)SPD and N¹,N⁴,N⁸-tri(ethyl)SPD], which differed in the number and/or position of the ethyl substitution (the alkyl series); three N-acetyl-SPD derivatives (N¹-acetyl-SPD, N⁴-acetyl-SPD, and N⁸-acetyl-SPD), which were comparable to the N-ethyl-SPD derivatives but not protonated at the substituted amine (the acyl series); three aliphatic analogs [nor-SPD, homo-SPD, and N¹,N⁹-bis(ethyl)homo-SPD], which differed in the interamine carbon chain length (homolog series), and 1,8-diaminooctane, which was comparable in overall chain length to SPD but lacked a central nitrogen. By comparing the relative abilities of the various analogs and SPD to modulate DNA structural transitions, it is possible to gain insight into the relative significance of the number and location of protonated amines (acyl series), the number and location of steric groups (alkyl series), aliphatic chain length (homolog series), and the central amine (1,8-diaminooctane) as determinants of SPD–DNA interactions. The B-to-Z conformational transition was facilitated to a midpoint by 2.4 μM SPD under conditions of low (i.e., 11 mM Na⁺) ionic strength. The phenomenon was affected most significantly by the number of protonated amines followed in rank order by location of the protonated amines, number of steric groups (bulk), steric group location, and aliphatic chain length. Stabilization of DNA to thermal melting was also most affected by the number of protonated amines followed by aliphatic chain length, number of steric groups, and location of protonated amines. In general, substitutions at the central (N⁴) amine of SPD exerted a significant influence on the B-to-Z transition but not on thermal melting.     

Antineoplastic and antiherpetic activity of spermidine catecholamide iron chelators

A series of iron chelating agents including the bacterial siderophores, parabactin and bis-N1,N8(2,3 dihydroxybenzoyl )spermidine, and four related compounds were synthesized and tested biologically. They were found: (a) to inhibit growth of cultured L1210 leukemia cells at IC50 values of 2-14 microM, (b) to inhibit replication of the DNA virus, herpes simplex type I, in monkey kidney cells at IC50 values of 0.4 microM ( parabactin ) to 55 microM, and (c) to be inactive against the RNA virus, vesicular stomatitis, at concentrations up to 1 mM. All effects were fully preventable by exogenous Fe (III). The activities correlated generally with the iron formation constants (10(36) to 10(48) moles/1) and more specifically with the lipophilicity of the compounds. The data suggest inhibition of DNA (but not RNA) synthesis by interference with the iron-containing enzyme, ribonucleotide reductase.     

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides,metabolically versatile members of ‘Proteobacteria’ from aquatic and soil habitats

RubisCO, the CO₂ fixing enzyme of the Calvin–Benson–Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes ¹²CO₂ faster than ¹³CO₂ resulting in ¹³C-depleted biomass, enabling the use of δ¹³C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ¹³C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the ‘Proteobacteria’ Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ¹³C values in nature.     

Receptor sites on human prostate tissue for prostaglandin F2 alpha.

The binding of high specific activity ³H-prostaglandin F₂ alpha to membranes derived from human prostate tissue is described. Binding is specific for PGF₂ alpha and is readily displaced by cold PGF₂ alpha. The influence of testosterone and lactogen on the binding of the prostaglandin is investigated. Testosterone appears to enhance binding while the binding observed in the presence of lactogen is difficult to interpret.     

Carbamoylcholine chloride induces a rapid increase in IL6 in the nasal cavity of C57BL/6 mice

Mice are widely used as models to study the roles of chemokines and cytokines in immune and inflammatory responses. In our work to determine the basal levels of cytokines in saliva, nasal wash fluid (NWF), bronchoalveolar lavage fluid (BALF), and serum of mice, we found that injection of carbamoylcholine chloride, used to stimulate saliva production, induced variations in the interleukin (IL) 6 levels of NWF and BALF supernatants. To characterize this response, C57BL/6 mice were given 10 microg carbamoylcholine chloride intraperitoneally and euthanized at 0, 1, 3, 6, 12, 24, 48, 72, and 96 h after injection. IL6 was increased in NWF supernatants by 2 to 3 h, remained elevated for 24 h, and declined by 48 h after injection. To determine whether carbamoylcholine chloride increased Th1 cytokine (IL2, IL12[p70], and interferon gamma), Th2 cytokine (IL4, IL5, and IL10), granulocyte-macrophage colony-stimulating factor (GM-CSF), or proinflammatory cytokine (IL1beta, tumor necrosis factor alpha, and IL6 in saliva and serum) levels, mice were given 10 microg carbamoylcholine chloride and euthanized. In 47 mice, all cytokine levels in saliva supernatants, NWF supernatants, BALF supernatants, and serum were within normal reported levels (range, 1 to 364 pg/ml); in the serum of the remaining 3 mice, GM-CSF, IL1beta, and IL2 levels were increased. In summary, carbamoylcholine chloride induces a rapid, elevated IL6 response in the nasal cavity and respiratory tract of mice but does not alter the levels of other Th1, Th2, or proinflammatory cytokines.     

HIV-1 gp120 vaccine induces affinity maturation in both new and persistent antibody clonal lineages

Most antibodies that broadly neutralize HIV-1 are highly somatically mutated in antibody clonal lineages that persist over time. Here, we describe the analysis of human antibodies induced during an HIV-1 vaccine trial (GSK PRO HIV-002) that used the clade B envelope (Env) gp120 of clone W6.1D (gp120_W6.1D). Using dual-color antigen-specific sorting, we isolated Env-specific human monoclonal antibodies (MAbs) and studied the clonal persistence of antibodies in the setting of HIV-1 Env vaccination. We found evidence of V_H somatic mutation induced by the vaccine but only to a modest level (3.8% ± 0.5%; range 0 to 8.2%). Analysis of 34 HIV-1-reactive MAbs recovered over four immunizations revealed evidence of both sequential recruitment of naïve B cells and restimulation of previously recruited memory B cells. These recombinant antibodies recapitulated the anti-HIV-1 activity of participant serum including pseudovirus neutralization and antibody-dependent cell-mediated cytotoxicity (ADCC). One antibody (3491) demonstrated a change in specificity following somatic mutation with binding of the inferred unmutated ancestor to a linear C2 peptide while the mutated antibody reacted only with a conformational epitope in gp120 Env. Thus, gp120_W6.1D was strongly immunogenic but over four immunizations induced levels of affinity maturation below that of broadly neutralizing MAbs. Improved vaccination strategies will be needed to drive persistent stimulation of antibody clonal lineages to induce affinity maturation that results in highly mutated HIV-1 Env-reactive antibodies.     

Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.     

Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ��

A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4–26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primer-templates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650–26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.Herpes simplex virus 1 (HSV-1)² encodes seven proteins essential for replicating its double-stranded DNA genome; five of these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products) and the heterodimeric polymerase (UL30-UL42 gene products) (1, 2). The helicase-primase unwinds the DNA at the replication fork and generates single-stranded DNA for both leading and lagging strand synthesis. Primase synthesizes short RNA primers on the lagging strand that the polymerase presumably elongates using dNTPs (i.e. primase-coupled polymerase activity). These two protein complexes are thought to replicate the viral genome on both the leading and lagging strands (1, 2).Previous studies have focused on the helicase-primase and polymerase separately. The helicase-primase contains three subunits, UL5, UL52, and UL8 in a 1:1:1 ratio (3–5). The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like motifs, yet the minimal active complex that demonstrates either helicase or primase activities contains both UL5 and UL52 (6, 7). Although the UL8 subunit has no known catalytic activity, several functions have been proposed, including enhancing helicase and primase activities, enhancing primer synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA strands, and facilitating formation of the replisome (8–12). Although primase will synthesize short (2–3 nucleotides long) primers on a variety of template sequences, synthesis of longer primers up to 13 nucleotides long requires the template sequence, 3′-deoxyguanidine-pyrimidine-pyrimidine-5′ (13). Primase initiates synthesis at the first pyrimidine via the polymerization of two purine NTPs (13). Even after initiation at this sequence, however, the vast majority of products are only 2–3 nucleotides long (13, 14).The herpes polymerase consists of the UL30 subunit, which has polymerase and 3′ → 5′ exonuclease activities (1, 2), and the UL42 subunit, which serves as a processivity factor (15–17). Unlike most processivity factors that encircle the DNA, the UL42 protein binds double-stranded DNA and thus directly tethers the polymerase to the DNA (18). Using pre-existing DNA primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42) incorporates dNTPs at a rate of 150 s^–1, a rate much faster than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01 s^–1) (19, 20).We examined primase-coupled polymerase activity by the herpes primase and polymerase complexes. Although herpes primase synthesizes RNA primers 2–13 nucleotides long, the polymerase only effectively elongates those at least 8 nucleotides long. Surprisingly, the polymerase elongated only a small fraction of the primase-synthesized primers (<1–2%), likely because of the polymerase elongating RNA primer-templates much less efficiently than DNA primer-templates. In contrast, human DNA polymerase α (pol α) elongated the herpes primase-synthesized primers very efficiently. The biological significance of these data is discussed.