Photodynamic herbicides: 1. Concept and phenomenology

A new approach to the design of conceptually and phenomenologically new herbicides is described. It involves the joint utilization of tetrapyrrole precursors, such as δ-aminolaevulinic acid (a biodegradable amino acid) and activators of the chlorophyll biosynthetic pathway, such as 2,2′-dipyridyl, in order to induce treated plants to biosynthesize and accumulate massive amounts of tetrapyrrole intermediates of the chlorophyll biosynthetic pathway in the dark (i.e. at night). During the subsequent light period (daylight) the accumulated tetrapyrroles act as potent photodynamic sensitiziers, which in turn result in the death of susceptible plants in a matter of hours. We have therefore proposed to name herbicides that act via this mechanism as photodynamic herbicides, or more pictorially as laser herbicides. From a limited survey of agricultural plant and weed species it appears that photodynamic herbicides exhibit a very pronounced organ, age and species-dependent selectivity. For example, dicotyledonous weeds such as mustard, red-root pigweed, common purslane and lambsquarter are very susceptible while monocotyledonous plants such as corn, wheat, barley and oats are not. The biochemical basis of this selectivity seems to lie, among other things, in the rates of tetrapyrrole turnover and in a differential enhancement by the applied chemicals of the monovinyl and divinyl tetrapyrrole biosynthetic pathways in the various species. A survey of various groups of chemicals (herbicides and other selected biochemicals) that are likely to exhibit photodynamic herbicidal properties is currently under investigation.     

Heat shock proteins: A dual carrier-adjuvant for an anti-drug vaccine against heroin

Heroin is a highly abused opioid that has reached epidemic status within the United States. Yet, existing therapies to treat addiction are inadequate and frequently result into rates of high recidivism. Vaccination against heroin offers a promising alternative therapeutic option but requires further development to enhance the vaccine’s performance. Hsp70 is a conserved protein with known immunomodulatory properties and is considered an excellent immunodominant antigen. Within an antidrug vaccine context, we envisioned Hsp70 as a potential dual carrier-adjuvant, wherein immunogenicity would be increased by co-localization of adjuvant and antigenic drug hapten. Recombinant Mycobacterium tuberculosis Hsp70 was appended with heroin haptens and the resulting immunoconjugate granted anti-heroin antibody production and blunted heroin-induced antinociception. Moreover, Hsp70 as a carrier protein surpassed our benchmark Her-KLH cocktail through antibody-mediated blockade of 6-acetylmorphine, the main mediator of heroin’s psychoactivity. The work presents a new avenue for exploration in the use of hapten-Hsp70 conjugates to elicit anti-drug immune responses.     

The discovery of benzoxazine sulfonamide inhibitors of NaV1.7: Tools that bridge efficacy and target engagement

The voltage-gated sodium channel Na_V1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of Na_V1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat Na_V1.7 versus human Na_V1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse Na_V1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.     

Impairment of the photosynthetic apparatus by oxidative stress induced by photosensitization reaction of protoporphyrin IX

Treatment with the herbicide acifluorfen-sodium (AF-Na), an inhibitor of protoporphyrinogen oxidase, caused an accumulation of protoporphyrin IX (Proto IX) , light-induced necrotic spots on the cucumber cotyledon within 12-24 h, and photobleaching after 48-72 h of light exposure. Proto IX-sensitized and singlet oxygen (¹O₂)-mediated oxidative stress caused by AF-Na treatment impaired photosystem I (PSI), photosystem II (PSII) and whole chain electron transport reactions. As compared to controls, the F_v/F_m (variable to maximal chlorophyll a fluorescence) ratio of treated samples was reduced. The PSII electron donor NH₂OH failed to restore the F_v/F_m ratio suggesting that the reduction of F_v/F_m reflects the loss of reaction center functions. This explanation is further supported by the practically near-similar loss of PSI and PSII activities. As revealed from the light saturation curve (rate of oxygen evolution as a function of light intensity), the reduction of PSII activity was both due to the reduction in the quantum yield at limiting light intensities and impairment of light-saturated electron transport. In treated cotyledons both the Q (due to recombination of Q_A⁻ with S₂) and B (due to recombination of Q_B⁻ with S₂/S₃) band of thermoluminescence decreased by 50% suggesting a loss of active PSII reaction centers. In both the control and treated samples, the thermoluminescence yield of B band exhibited a periodicity of 4 suggesting normal functioning of the S states in centers that were still active. The low temperature (77 K) fluorescence emission spectra revealed that the F₆₉₅ band (that originates in CP-47) increased probably due to reduced energy transfer from the CP47 to the reaction center. These demonstrated an overall damage to the PSI and PSII reaction centers by ¹O₂ produced in response to photosensitization reaction of protoporphyrin IX in AF-Na-treated cucumber seedlings.     

Discovery of two novel branched peptidomimetics containing endomorphin-2 and RF9 pharmacophores: Synthesis and neuropharmacological evaluation

It is well known that opioid analgesics produce side effects including tolerance and constipation. Since neuropeptide FF (NPFF) receptor antagonists reversed opioid-induced hyperalgesia and analgesic tolerance, the present work was performed to synthetize two branched peptidomimetics, EKR and RKE, containing the opioid peptide endomorphin-2 (EM-2) and the NPFF receptor antagonist RF9. Our data obtained from the in vitro cyclic adenosine monophosphate experiment demonstrated that EKR functioned as a mixed mu-, delta-opioid receptors agonist and NPFF₁ receptor antagonist/NPFF₂ receptor partial agonist, whereas RKE acted as a multi-functional peptidomimetic with the mu-opioid agonism and the NPFF₁ antagonism/NPFF₂ partial agonism. Furthermore, EKR and RKE completely blocked the NPFF₂ receptor-mediated neurite outgrowth of Neuro 2A cells. In vivo antinociception studies found that supraspinal administration of EKR and RKE dose-dependently produced potent antinociception via the mu-opioid receptor in the tail-flick test. In carrageenan inflammatory pain model, spinal administration of EKR and RKE induced dose-related analgesia, which was significantly reduced by the opioid antagonist naloxone and the NPFF antagonist RF9. Notably, compared with morphine, intracerebroventricular repeated administration of EKR and RKE maintained prolonged antinociceptive effectiveness. In addition, at the antinociceptive doses, these two branched peptidomimetics did not significantly inhibit gastrointestinal transit. Taken together, the present work suggest that EKR and RKE behave as multi-functional ligands with the opioid agonism and the NPFF₁ antagonism/NPFF₂ partial agonism, and produce prolonged antinociception with limited side effects. Moreover, our results imply that EKR and RKE might be interesting pharmacological tools for further investigating the biological function of the NPFF and opioid systems.     

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.     

Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.     

Use of replicates in statistical analyses in papers submitted for publication in Animal Feed Science and Technology

This editorial is an annex, essentially an amplification of earlier Animal Feed Science and Technology (AFST) editorials which focused on analytical and statistical issues of papers submitted for publication in AFST. This amplification is needed because, since publication of those editorials, there has been a sharp increase in papers submitted to AFST (particularly in vitro gas, ruminal in sacco, continuous culture fermenter and mini-silo studies), in which there have been substantive disagreements among authors, reviewers and editors as to what, exactly, constitutes acceptable statistical replicates as opposed to pseudo replicates. In this editorial, the Co-Editors in Chief of AFST provide clarification of their view relative to use of analytical observations in statistical analyses in papers submitted for consideration for publication in AFST. If the objective is to compare feeds and from the results, make inferences to populations, only multiple samples of each feed represent an acceptable feed base. This suggests that means comparisons based on repeated assays of the same sample, whether by chemical, physical or microbiological methods, will almost certainly be rejected by the CEIC.     

Unique utilization of a phosphoprotein phosphatase fold by a mammalian phosphodiesterase associated with WAGR syndrome

Metallophosphoesterase-domain-containing protein 2 (MPPED2) is a highly evolutionarily conserved protein with orthologs found from worms to humans. The human MPPED2 gene is found in a region of chromosome 11 that is deleted in patients with WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, and MPPED2 may function as a tumor suppressor. However, the precise cellular roles of MPPED2 are unknown, and its low phosphodiesterase activity suggests that substrate hydrolysis may not be its prime function. We present here the structures of MPPED2 and two mutants, which show that the poor activity of MPPED2 is not only a consequence of the substitution of an active-site histidine residue by glycine but also due to binding of AMP or GMP to the active site. This feature, enhanced by structural elements of the protein, allows MPPED2 to utilize the conserved phosphoprotein-phosphatase-like fold in a unique manner, ensuring that its enzymatic activity can be combined with a possible role as a scaffolding or adaptor protein.