Warfare Nerve Agents and Paraoxonase-1 as a Potential Prophylactic Therapy against Intoxication.

Nerve agents are a class of lethal neurotoxic chemicals used in chemical warfare. In this review, we have briefly discussed a brief history of chemical warfare, followed by an exploration of the historical context surrounding nerve agents. The article explores the classification of these agents, their contemporary uses, their toxicity mechanisms, and the disadvantages of the current treatment options for nerve agent poisoning. It then discusses the possible application of enzymes as prophylactics against nerve agent poisoning, outlining the benefits and drawbacks of paraoxonase-1. Finally, the current studies on paraoxonase-1 are reviewed, highlighting that several challenges need to be addressed in the use of paraoxonase-1 in the actual field and that its potential as a prophylactic antidote against nerve agent poisoning needs to be evaluated. The literature used in this manuscript was searched using various electronic databases, such as PubMed, Google Scholar, Web of Science, Elsevier, Springer, ACS, Google Patent, and books using the keywords chemical warfare agent, Butyrylcholinesterase, enzyme, nerve agent, prophylactic, and paraoxonase- 1, with the time scale for the analysis of articles between 1960 to 2023, respectively. The study has suggested that concerted efforts by researchers and agencies must be made to develop effective countermeasures against NA poisoning and that PON1 has suitable properties for the development of efficient prophylaxis against NA poisoning.

Is Human Paraoxonase 1 the Saviour Against the Persistent Threat of Organophosphorus Nerve Agents?

Nerve agents have been used extensively in chemical warfare in the past. However, recent use of Novichok agents have reignited the debate on the threat posed by Organophosphorus Nerve Agents (OPNAs). The currently available therapy for OPNA toxicity is only symptomatic and is potentially ineffective in neutralizing OPNAs. Hence, there is a dire need to develop a prophylactic therapy for counteracting OPNA toxicity. In this regard, human paraoxonase 1 has emerged as the enzyme of choice. In this review, we have focussed upon the recent and past events of OPNA use, their mechanism of action and toxicity. Further, we have emphasized upon the potential of enzyme based therapy and the various advances in the development of paraoxonase 1 as a countermeasure for OPNA poisoning. Finally, we have elaborated the shortcomings of paraoxonase 1 and the work that needs to be undertaken in order to develop human paraoxonase 1 as a prophylactic against OPNA poisoning.

Protein Chimerization: A New Frontier for Engineering Protein Therapeutics with Improved Pharmacokinetics.

With the advancement of medicine, the utility of protein therapeutics is increasing exponentially. However, a significant number of protein therapeutics suffer from grave limitations, which include their subpar pharmacokinetics. In this study, we have reviewed the emerging field of protein chimerization for improving the short circulatory half-life of protein therapeutics. We have discussed various aspects of protein therapeutics aiming at their mechanism of clearance and various approaches used to increase their short circulatory half-life with principal focus on the concept of chimerization. Furthermore, we have comprehensively reviewed various components of chimera, such as half-life extension partners and linkers, their shortcomings, and prospective work to be undertaken for developing effective chimeric protein therapeutics.

Organophosphate-Hydrolyzing Enzymes as First-Line of Defence Against Nerve Agent-Poisoning: Perspectives and the Road Ahead.

Nerve agents (NAs) are extremely neurotoxic synthetic organophosphate (OP) compounds exploited as weapons of mass destruction in terrorist attacks and chemical warfare. Considering the current world scenario, there is a persistent threat of NA-exposure to military personals and civilians. Various prophylactic and post-exposure treatments (such as atropine and oximes) available currently for NA-poisoning are inadequate and unsatisfactory and suffer from severe limitations. Hence, developing safe and effective treatment(s) against NA-poisoning is a critical necessity. With regards to counteracting NA-toxicity, the OP-hydrolyzing enzymes (OPHEs), which can hydrolyze and inactivate a variety of NAs, have emerged as promising candidates for the development of prophylactic therapy against NA-poisoning. However, there are many hurdles to be crossed before these enzymes can be brought to therapeutic use in humans. In this article, we have reviewed the various advancements in the field of development of OPHEs as prophylactic against NA-poisoning. The article majorly focuses on the toxic effects of NAs, various available therapies to counteract NA poisoning, the current status of OPHEs and attempts made to improve the various properties of these enzymes. Further, we have also briefly discussed about the prospective work that is needed to be undertaken for developing these OPHEs into those suitable for use in humans.

Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192.

Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1.

Improving storage stability of recombinant organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) is a ∼38kDa enzyme encoded by opd gene of Flavobacterium sp. The enzyme can hydrolyze and inactivate variety of organophosphate (OP)-compounds, including chemical warfare nerve agents. Thus, OPH is a strong candidate for the development of therapeutic intervention against OP-poisoning in humans and other animals. It is also a promising bio-decontaminating agent for clean-up of OP-contaminated objects and areas. For successful commercial application, long-term storage stability of purified OPH enzyme is important. In this study we have cloned and expressed recombinant OPH (r-OPH) in Escherichia coli and the effect of different excipients on the long-term storage stability of purified enzyme was analyzed. The enzyme was stored in either aqueous solution or in lyophilized form at 25°C for 60days in the presence or absence of different excipients and the stability of the enzyme was determined by monitoring the paraoxon-hydrolyzing activity. Our results suggest that, (a) maltose, trehalose, arginine and proline were most effective in stabilizing the enzyme when stored in aqueous buffer at 25°C, and (b) maltose, trehalose, and mannose exerted maximum stabilization effect when the enzyme was stored in lyophilized form at 25°C for 60days. The study shows that common excipients can be used to stabilize purified OPH enzyme in order to store it for long period of time under different storage conditions. The results of this study can be used to develop formulation(s) of OPH enzyme for commercial use.

Crouzons syndrome: a case report with review of literature.

Louis Edouard Octave Crouzon, a French neurologist, in 1912, described the hereditary syndrome of craniofacial synostosis in a mother and son. He described the triad as skull deformities, facial anamolies and exopthalmos now known as Crouzon syndrome (CS). CS accounts for about 4.8% of all cases of craniosynostosis. We report a case of CS in 4 year old girl with characteristic features of cranial deformity, maxillary hypoplasia, cleft palate and exopthalmos.

Human ARHGDIG, a GDP-dissociation inhibitor for Rho proteins: genomic structure, sequence, expression analysis, and mapping to chromosome 16p13.3.

GDP-dissociation inhibitors (GDIs) play a primary role in modulating the activity of GTPases. We recently reported the identification of a new GDI for the Rho-related GTPases named RhoGDIgamma. This gene is now designated ARHGDIG by HUGO. Here, in a detailed analysis of tissue expression of ARHGDIG, we observe high levels in the entire brain, with regional variations. The mRNA is also present at high levels in kidney and pancreas and at moderate levels in spinal cord, stomach, and pituitary gland. In other tissues examined, the mRNA levels are very low (lung, trachea, small intestine, colon, placenta) or undetectable. RT-PCR analysis of total RNA isolated from exocrine pancreas and islets shows that the gene is expressed in both tissues. We also report the genomic structure of ARHGDIG. The gene spans over 4 kb and is organized into six exons and five introns. The upstream region lacks a canonical TATA box and contains several putative binding sites for ubiquitous and tissue-specific factors active in central nervous system development. Using FISH, we have mapped the gene to chromosome band 16p13.3. This band is rich in deletion mutants of genes involved in several human diseases, notably polycystic kidney disease, alpha-thalassemia, tuberous sclerosis, mental retardation, and cancer. The promoter structure and the chromosomal location of RhoGDIgamma suggest its importance and underscore the need for further investigation into its biology.

Mechanisms of indirect allorecognition: characterization of MHC class II allopeptide-specific T helper cell clones from animals undergoing acute allograft rejection.

BACKGROUND: Recent evidence indicates that T cells primed via the indirect pathway of allorecognition play an important role in allograft rejection, although the effector mechanisms remain unknown. The purpose of this study was to characterize and study the in vivo function of self-restricted MHC allopeptide-specific T-cell clones generated from animals undergoing allograft rejection. METHODS AND RESULTS: We generated self-restricted class II MHC allopeptide-specific T-cell clones from the spleen and kidney of Lewis (LEW; RT1l) rats undergoing acute rejection of MHC-incompatible Wistar Furth (WF; RT1u) renal allografts. RT1.Du/beta20-44 peptide-specific CD4+ T helper 1 clones from the spleen and kidney of rejecting animals expressed a restricted T cell receptor (TCR) Vbeta repertoire: Vbeta4, 8.2, or 9. In comparison, clones generated from RT1.Dubeta20-44 immunized LEW rats all expressed TCR Vbeta9. The amino acid sequence of RT1.Dl (LEW) and RT1.Du (WF) residues 20-44 differ only at positions 30 and 38. T-cell clones expressing TCR Vbeta9 preferentially proliferated to the peptide fragment RT1.Dubeta20-33. T-cell clones expressing TCR Vbeta4 proliferated weakly to peptide fragments RT1.Dubeta20-33 and 31-44, whereas those expressing TCR Vbeta8.2 proliferated preferentially to the peptide fragment 31-44. Adoptive transfer of T-cell clones expressing TCR Vbeta9 or Vbeta8.2, but not Vbeta4, to naive LEW animals elicited significant delayed-type hypersensitivity responses after challenge with the RT1.Dubeta20-44 peptide or allogeneic WF (RT1u) splenocytes. CONCLUSION: This is the first report on the cellular, molecular, and functional characterization of self-restricted MHC allopeptide-specific T-cell clones from animals undergoing acute rejection. Our data provide support for a biologically significant role of indirect allorecognition in allograft rejection.

Striking augmentation of hematopoietic cell chimerism in noncytoablated allogeneic bone marrow recipients by FLT3 ligand and tacrolimus.

The influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and the recently identified hematopoietic stem-progenitor cell mobilizing factor flt3 ligand (FL) on donor leukocyte microchimerism in noncytodepleted recipients of allogeneic bone marrow (BM) was compared. B10 mice (H2b) given 50x10(6) allogeneic (B10.BR [H2k]) BM cells also received either GM-CSF (4 microg/day s.c.), FL (10 microg/day i.p.), or no cytokine, with or without concomitant tacrolimus (formerly FK506; 2 mg/kg) from day 0. Chimerism was quantitated in the spleen 7 days after transplantation by both polymerase chain reaction (donor DNA [major histocompatibility complex class II; I-E(k)]) and immunohistochemical (donor [I-E(k)+] cell) analyses. Whereas GM-CSF alone significantly augmented (fivefold) the level of donor DNA in recipients' spleens, FL alone caused a significant (60%) reduction. Donor DNA was increased 10-fold by tacrolimus alone, whereas coadministration of GM-CSF and tacrolimus resulted in a greater than additive effect (28-fold increase). A much more striking effect was observed with FL + tacrolimus (>125-fold increase in donor DNA compared with BM alone). These findings were reflected in the relative numbers of donor major histocompatibility complex class II+ cells (many resembling dendritic cells) detected in spleens, although quantitative differences among the groups were less pronounced. Evaluation of cytotoxic T lymphocyte generation by BM recipients' spleen cells revealed that FL alone augmented antidonor immunity and that this was reversed by tacrolimus. Thus, although FL may potentiate antidonor reactivity in nonimmunosuppressed, allogeneic BM recipients, it exhibits potent chimerism-enhancing activity when coadministered with recipient immunosuppressive therapy.

Follicle cell calmodulin in Blattella germanica: transcript accumulation during vitellogenesis is regulated by juvenile hormone.

There is abundant calmodulin (CaM) in the oocytes and eggs of B. germanica. Whether oocytes accumulate CaM for immediate use or use at a later stage in their development is still unknown. We show that isolated follicle cells accumulate more CaM transcripts per unit RNA than any other control tissue. CaM transcript increases exponentially 4800-fold in follicles during the 96-hr vitellogenic period in the absence of cell division. This includes a 32-fold increase in total follicle RNA during the period and an 150-fold increase in relative titer of the CaM transcript. In comparison, levels of actin transcripts increase exponentially 1200-fold during the same developmental period. On the other hand fat body tissue shows little relative increase of CaM transcripts despite a 4-fold increase in total RNA over the 4-day developmental period. Both the CaM and actin transcripts are more highly concentrated in the Day 4 follicle cell layer, being found in 84- and 33-fold greater titer, respectively, than in fat body RNA. Deprivation of juvenile hormone (JH), by head ligation, not only causes atresia of the follicles, but also reduces accumulated CaM transcripts. Reestablishing JH titer by injection allows a selected population of follicles to develop to full size and also reinstates CaM transcript levels above that of unligated controls within 24 hr.