Coexpression of molecular chaperone enhances activity and export of organophosphorus hydrolase in Escherichia coli

Periplasmic secretion has been used in attempts to construct an efficient whole‐cell biocatalyst with greatly reduced diffusion limitations. Previously, we developed recombinant Escherichia coli that express organophosphorus hydrolase (OPH) in the periplasmic space using the twin‐arginine translocation (Tat) pathway to degrade environmental toxic organophosphate compounds. This system has the advantage of secreting protein into the periplasm after folding in the cytoplasm. However, when OPH was expressed with a Tat signal sequence in E. coli, we found that the predominant OPH was an insoluble premature form in the cytoplasm, and thus, the whole‐cell OPH activity was significantly lower than its cell lysate activity. In this work, we, for the first time, used a molecular chaperone coexpression strategy to enhance whole‐cell OPH activity by improving the periplasmic translocation of soluble OPH. We found that the effect of GroEL‐GroES (GroEL/ES) assistance on the periplasmic localization of OPH was secretory pathway dependent. We observed a significant increase in the amount of soluble mature OPH when cytoplasmic GroEL/ES was expressed; this increase in the amount of mature OPH might be due to enhanced OPH folding in the cytoplasm. Importantly, the whole‐cell OPH activity of the chaperone–coexpressing cells was ～5.5‐fold greater at 12 h after induction than that of cells that did not express the chaperone as a result of significant Tat‐based periplasmic translocation of OPH in the chaperone–coexpressing cells. Collectively, these data suggest that molecular chaperones significantly enhance the whole‐cell activity of periplasmic OPH‐secreting cells, yielding an effective whole‐cell biocatalyst system with highly reduced diffusion limitations. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 925–930, 2012     

TatD is a central component of a Tat translocon‐initiated quality control system for exported FeS proteins in Escherichia coli

Bacterial Tat systems export folded proteins, including FeS proteins such as NrfC and NapG, which acquire their cofactors before translocation. NrfC and NapG are proofread by the Tat pathway, and misfolded examples are degraded after interaction with the translocon. Here, we identify TatD as a crucial component of this quality control system in Escherichia coli. NrfC/NapG variants lacking FeS centres are rapidly degraded in wild‐type cells but stable in a ΔtatD strain. The precursor of another substrate, FhuD, is also transiently detected in wild‐type cells but stable in the ΔtatD strain. Surprisingly, these substrates are stable in ΔtatD cells that overexpress TatD, and export of the non‐mutated precursors is inhibited. We propose that TatD is part of a quality control system that is intimately linked to the Tat export pathway, and that the overexpression of TatD leads to an imbalance between the two systems such that both Tat‐initiated turnover and export are prevented.     

Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport

The thylakoid DeltapH-dependent pathway transports folded proteins with twin arginine-containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an approximately 700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC-Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of DeltapH. These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.     

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex

The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.     

Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.     

Discovery of amyloid‐beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility

Genetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid β peptide (Aβ), the so‐called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Aβ from its precursor (β‐ and γ‐secretases) or interrupt and/or reverse Aβ aggregation. To identify potent inhibitors of Aβ aggregation, we have developed a high‐throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42‐residue AD pathogenesis effecter Aβ42. Specifically, a tripartite fusion between the Tat‐dependent export signal ssTorA, the Aβ42 peptide and the β‐lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Aβ42 moiety. Here, we reasoned that small, cell‐permeable molecules that inhibited Aβ42 aggregation would render the ssTorA‐Aβ42‐Bla chimera competent for Tat export to the periplasm where Bla is active against β‐lactam antibiotics such as ampicillin. Using a fluorescence‐based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell‐permeable compounds that promoted efficient Tat‐dependent export of ssTorA‐Aβ42‐Bla. Each of these was subsequently shown to be a bona fide inhibitor of Aβ42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.     

Multiple precursor proteins bind individual Tat receptor complexes and are collectively transported

The thylakoid t win a rginine protein t ranslocation (Tat) system is thought to have a multivalent receptor complex with each cpTatC‐Hcf106 pair constituting a signal peptide‐binding unit. Conceptual models suggest that translocation of individual precursor proteins occurs upon assembly of a Tha4 oligomer with a precursor‐occupied cpTatC‐Hcf106. However, results reported here reveal that multiple precursor proteins bound to a single receptor complex can be transported together. Precursor proteins that contain one or two cysteine residues readily formed intermolecular disulphide bonds upon binding to the receptor complex, resulting in dimeric and tetrameric precursor proteins. Three lines of evidence indicate that all members of precursor oligomers were specifically bound to a receptor unit. Blue native–polyacrylamide gel electrophoresis analysis showed that oligomers were present on individual receptor complexes rather than bridging two or more receptor complexes. Upon energizing the membrane, the dimeric and tetrameric precursors were transported across the membrane with efficiencies comparable with that of monomeric precursors. These results imply a novel aspect of Tat systems, whereby multiple precursor‐binding sites can act in concert to transport an interlinked oligo‐precursor protein.     

Component specificity for the thylakoidal Sec and Delta pH-dependent protein transport pathways.

Prokaryotes and prokaryote-derived thylakoid membranes of chloroplasts share multiple, evolutionarily conserved pathways for protein export. These include the Sec, signal recognition particle (SRP), and Delta pH/Tat systems. Little is known regarding the thylakoid membrane components involved in these pathways. We isolated a cDNA clone to a novel component of the Delta pH pathway, Tha4, and prepared antibodies against pea Tha4, against maize Hcf106, a protein implicated in Delta pH pathway transport by genetic studies, and against cpSecY, the thylakoid homologue of the bacterial SecY translocon protein. These components were localized to the nonappressed thylakoid membranes. Tha4 and Hcf106 were present in approximately 10-fold excess over active translocation sites. Antibodies to either Tha4 or Hcf106 inhibited translocation of four known Delta pH pathway substrate proteins, but not of Sec pathway or SRP pathway substrates. This suggests that Tha4 and Hcf106 operate either in series or as subunits of a heteromultimeric complex. cpSecY antibodies inhibited translocation of Sec pathway substrates but not of Delta pH or SRP pathway substrates. These studies provide the first biochemical evidence that Tha4 and Hcf106 are specific components of the Delta pH pathway and provide one line of evidence that cpSecY is used specifically by the Sec pathway.     

Photoacoustic imaging of the human placental vasculature

Minimally invasive fetal interventions require accurate imaging from inside the uterine cavity. Twin‐to‐twin transfusion syndrome (TTTS), a condition considered in this study, occurs from abnormal vascular anastomoses in the placenta that allow blood to flow unevenly between the fetuses. Currently, TTTS is treated fetoscopically by identifying the anastomosing vessels, and then performing laser photocoagulation. However, white light fetoscopy provides limited visibility of placental vasculature, which can lead to missed anastomoses or incomplete photocoagulation. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, two PA systems were used to visualize chorionic (fetal) superficial and subsurface vasculature in human placentas. The first system comprised an optical parametric oscillator for PA excitation and a 2D Fabry‐Pérot cavity ultrasound sensor; the second, light emitting diode arrays and a 1D clinical linear‐array ultrasound imaging probe. Volumetric photoacoustic images were acquired from ex vivo normal term and TTTS‐treated placentas. It was shown that superficial and subsurface branching blood vessels could be visualized to depths of approximately 7 mm, and that ablated tissue yielded negative image contrast. This study demonstrated the strong potential of PA imaging to guide minimally invasive fetal therapies.      

Mining mammalian genomes for folding competent proteins using Tat‐dependent genetic selection in Escherichia coli

Recombinant expression of eukaryotic proteins in Escherichia coli is often limited by poor folding and solubility. To address this problem, we employed a recently developed genetic selection for protein folding and solubility based on the bacterial twin‐arginine translocation (Tat) pathway to rapidly identify properly folded recombinant proteins or soluble protein domains of mammalian origin. The coding sequences for 29 different mammalian polypeptides were cloned as sandwich fusions between an N‐terminal Tat export signal and a C‐terminal selectable marker, namely β‐lactamase. Hence, expression of the selectable marker and survival on selective media was linked to Tat export of the target mammalian protein. Since the folding quality control feature of the Tat pathway prevents export of misfolded proteins, only correctly folded fusion proteins reached the periplasm and conferred cell survival. In general, the ability to confer growth was found to relate closely to the solubility profile and molecular weight of the protein, although other features such as number of contiguous hydrophobic amino acids and cysteine content may also be important. These results highlight the capacity of Tat selection to reveal the folding potential of mammalian proteins and protein domains without the need for structural or functional information about the target protein.     

Localization and integration of thylakoid protein translocase subunit cpTatC

Thylakoid membranes have a unique complement of proteins, most of which are nuclear encoded synthesized in the cytosol, imported into the stroma and translocated into thylakoid membranes by specific thylakoid translocases. Known thylakoid translocases contain core multi-spanning, membrane-integrated subunits that are also nuclear-encoded and imported into chloroplasts before being integrated into thylakoid membranes. Thylakoid translocases play a central role in determining the composition of thylakoids, yet the manner by which the core translocase subunits are integrated into the membrane is not known. We used biochemical and genetic approaches to investigate the integration of the core subunit of the chloroplast Tat translocase, cpTatC, into thylakoid membranes. In vitro import assays show that cpTatC correctly localizes to thylakoids if imported into intact chloroplasts, but that it does not integrate into isolated thylakoids. In vitro transit peptide processing and chimeric precursor import experiments suggest that cpTatC possesses a stroma-targeting transit peptide. Import time-course and chase assays confirmed that cpTatC targets to thylakoids via a stromal intermediate, suggesting that it might integrate through one of the known thylakoid translocation pathways. However, chemical inhibitors to the cpSecA-cpSecY and cpTat pathways did not impede cpTatC localization to thylakoids when used in import assays. Analysis of membranes isolated from Arabidopsis thaliana mutants lacking cpSecY or Alb3 showed that neither is necessary for cpTatC membrane integration or assembly into the cpTat receptor complex. These data suggest the existence of another translocase, possibly one dedicated to the integration of chloroplast translocases.     

Effects of oral L-arginine supplementation on blood pressure and asymmetric dimethylarginine in stress-induced preeclamptic rats

This study was carried out to elucidate the role of asymmetric dimethylarginine (ADMA) and nitric oxide (NO) in preeclampsia development, and to investigate the effect of L-arginine supplementation in rats. Preeclampsia was induced in pregnant rats using a stress model. L-arginine was administered orally and ADMA, urinary nitrate, and protein levels were measured on the 20th day of pregnancy. Compared with the group of rats that are normally pregnant, the levels of blood pressure (BP), protein excretion, and ADMA were significantly increased in preeclampsia which returned to normal levels following the supplementation of L-arginine. Both group of rats had similar urine nitrate levels. Arginine-ADMA-NO pathway is affected in preeclampsia. L-arginine supplementation decreased hypertension (HT), proteinuria, and ADMA levels indicating that taking L-arginine may be beneficial in preeclampsia treatment.     

In vivo dissection of the Tat translocation pathway in Escherichia coli

The bacterial Tat pathway is capable of exporting folded proteins carrying a special twin arginine (RR) signal peptide. By using two in vivo reporter proteins, we assessed factors that affect Tat pathway transport. We observed that, like the intact RR signal peptide, those with a KR or RK substitution were still capable of mediating the translocation of the folded green fluorescent protein (GFP). However, the translocation efficiency decreased in the order of RR>KR>RK. The KK motif was unable to mediate GFP translocation. The translocation of the RR-GFP fusion required TatA, TatB and TatC proteins. By exploiting the periplasmic bactericidal property of colicin V (ColV), we constructed a translocation-suicide probe, RR-ColV. The translocation of RR-ColV fully inhibited the growth of wild-type Escherichia coli and those of the DeltatatD and DeltatatE mutants. In contrast, the deletion of the tatC gene blocked RR-ColV in the cytoplasm and this strain exhibited a normal growth phenotype. Interestingly, the growth of DeltatatA and tatB mutants was inhibited partially by RR-ColV. Moreover, KR, RK and KK motifs were capable of mediating the ColV translocation with a decreasing RR=KR>RK>KK efficiency. In addition to TatE and TatC proteins, either TatA or TatB was sufficient for the translocation of RR-ColV or KR-ColV. In contrast, TatA plus the conserved N-terminal domain of TatB were required to mediate the killing effect of ColV fused to the less-efficient RK signal peptide. Taken together, these results suggest that a fully efficient Tat pathway transport is determined by the sequence of the signal peptide, the composition of the Tat apparatus, and the intrinsic characteristics of exported proteins.     

l-Arginine hydrochloride increases the solubility of folded and unfolded recombinant plasminogen activator rPA

L ‐Arginine hydrochloride (L ‐ArgHCl) was found to be an effective enhancer for in vitro protein refolding more than two decades ago. A detailed understanding of the mechanism of action, by which L ‐ArgHCl as co‐solvent is capable to effectively suppress protein aggregation, while protein stability is preserved, has remained elusive. Concepts for the effects of co‐solvents, which have been established over the last decades, were found to be insufficient to completely explain the effects of L ‐ArgHCl on protein refolding. In this article, we present data, which clearly establish that L ‐ArgHCl acts on the equilibrium solubility of the native model protein recombinant plasminogen activator (rPA), while for S‐carboxymethylated rPA (IAA‐rPA) that served as a model protein for denatured protein states, equilibrium solubilities could not be obtained. Solid to solute free transfer energies for native rPA were lowered by up to 14 kJ mol^‐1 under the tested conditions. This finding is in marked contrast to a previously proposed model in which L ‐ArgHCl acts as a neutral crowder which exclusively has an influence on the stability of the transition state of aggregation. The effects on the apparent solubility of IAA‐rPA, as well as on the aggregation kinetics of all studied protein species, that were observed in the present work could tentatively be explained within the framework of a nucleation‐aggregation scheme, in which L ‐ArgHCl exerts a strong effect on the pre‐equilibria leading to formation of the aggregation seed.     

Reduced L‐arginine transport contributes to the pathogenesis of myocardial ischemia‐reperfusion injury

Myocardial injury due to ischemia‐reperfusion (I‐R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I‐R process induces a state of insufficient L ‐arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I‐R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia‐reoxygenation significantly decreased L ‐arginine uptake and NO production (42 ± 2% and 71 ± 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L ‐arginine transporter, CAT1, and NVCMs supplemented with L ‐arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia‐reoxygenation. In contrast, L ‐arginine deprived NVCM had significantly worsened responses to hypoxia‐reoxygenation. In isolated perfused mouse hearts, L ‐arginine infusion during reperfusion significantly improved left ventricular function after I‐R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L ‐arginine availability as a key factor in the pathogenesis of I‐R injury. Increasing L ‐arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I‐R. Restoration of L ‐arginine availability may therefore be a valuable strategy to ameliorate I‐R injury. J. Cell. Biochem. 108: 156–168, 2009. © 2009 Wiley‐Liss, Inc.     

精氨酸-胍基丁胺代谢的研究进展

近几年来,随着对精氨酸代谢的研究,发现除了存在精氨酸-一氧化氮代谢途径外,还存在着精氨酸一胍基丁胺代谢途径。该反应由精氨酸脱羧酶催化,在体内有广泛的分布。这两条代谢途径间存在着密切的相互调节关系。     

Isothiocyanate groups of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) inhibit cell penetration of octa‐arginine (R8)–fused peptides

Delivering biomolecules, such as antibodies, proteins, and peptides, to the cytosol is an important and challenging aspect of drug development and chemical biology. Polyarginine—a well‐known cell‐penetrating peptide (CPP)—is capable of exploiting its positive charge and guanidium groups to carry a fused cargo into the cytosol. However, the precise mechanism by which this occurs remains ambiguous. In the present study, we established a new method of quantitatively assessing cell penetration. The method involves inducing cell death by using a polyarginine (R8) to deliver a peptide—ie, mitochondrial targeting domain (MTD)—to the cytosol. We found that 4,4′‐diisothiocyanatostilbene‐2,2′‐di‐sulfonate (DIDS)—an anion channel blocker—inhibited the ability of octa‐arginine (R8)–fused MTD to penetrate cells. Other anion channel blockers did not inhibit the penetration of peptides fused with R8. Comparison of DIDS with other structurally similar chemicals revealed that the isothiocyanate group of DIDS may be primarily responsible for the inhibitory effect than its stilbene di‐sulfonate backbone. These results imply that the inhibitory effect of DIDS may not be derived from the interaction between stilbene di‐sulfonate and the anion channels, but from the interaction between the isothiocyanate groups and the cell membrane. Our new MTD method enables the quantitative assessment of cell penetration. Moreover, further studies on the inhibition of CPPs by DIDS may help clarify the mechanism by which penetration occurs and facilitate the design of new penetrative biomolecules.     

The mechanism and modes of inhibition of arginine kinase from the cockroach (Periplaneta americana)

The kinetic mechanism and evaluation of several potential inhibitors of purified arginine kinase from the cockroach (Periplanta americana) were investigated. This monomeric phosphagen kinase is important in maintaining ATP levels during the rapid energy demands of muscle required for contraction and motility. Analysis reveals the following dissociation constants (mM) for the binary complex: E.Arg P-->E+Arg P, K=1.0; E.Arg-->E+Arg, K=0.45; E.MgATP-->E+MgATP, K=0.17; E.MgADP-->E+MgADP, K=0.12; and the ternary complex: Arg P.E.MgADP-->E.MgADP+Arg P, K=0.94; Arg.E.MgATP-->E.MgATP+Arg, K=0.49; MgATP.Enz.Arg-->E.Arg+MgATP, K=0.14; MgADP.E.Arg P-->E.Arg P+MgADP, K=0.09. For a particular substrate, the ratio of the dissociation constants for the binary to ternary complex is close to one, indicating little, if any, cooperativity in substrate binding for the rapid equilibrium, random addition mechanism. The time course of the arginine kinase reaction exhibits a pronounced curvature, which, as described for enzyme from other sources, is attributed to formation of an inhibitory catalytic dead-end complex, MgADP.E.Arg. The curvature is accentuated by the addition of monovalent anions, including borate, thiocyanate, and, most notably, nitrite and nitrate. This effect is attributed to stabilization of the dead-end complex through formation of a transition state analog. However, the substantial decrease in initial velocity (92%) caused by nitrate is due to an additional inhibitory effect, further characterized as non-competitive inhibition (Ki=8.0 mM) with the substrate L-arginine. On the other hand, borate inhibition of the initial velocity is only 30% with significant subsequent curvature, suggesting that this anion functions as an inhibitor mainly by formation of a transition state analog. However, some component of the borate inhibition appears to be mediated by an apparent partial competitive inhibition with L-arginine. D-arginine is not a substrate for arginine kinase from the cockroach, but is an effective competitive inhibitor with a Ki=0.31 mM. L-Canavanine is a weak substrate for arginine kinase (Km=6.7 mM) with a Vmax for the pure enzyme that is approximately one-third that of L-arginine. However, initial velocity experiments of substrate mixtures suggest that competition between L-canavanine and L-arginine may not be a simple summation effect and may involve a structural modification. Sensitivity of arginine kinase activity to D-arginine as well as nitrate and borate anions, coupled with the fact that L-arginine is an essential amino acid for the cockroach, suggest that arginine kinase could be a useful chemotherapeutic target for the control of cockroach proliferation.     

The effect of L-arginine or L-citrulline supplementation on biochemical parameters and the vascular aortic wall in high-fat and high-cholesterol-fed rats

The aim of the present study is to investigate the potential role of L-arginine or L-citrulline in rats fed high-fat and high-cholesterol (HFC) diet. HFC feeding increased significantly serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, urea and all lipid profiles and decreased significantly serum high-density lipoprotein-cholesterol (HDL-c) and non significantly serum nitric oxide levels. L-arginine or L-citrulline administration reversed the increase in serum AST and ALT activities, urea and all lipid profiles. These effects were associated with a concomitant increase in HDL-c and nitric oxide levels. In general, rats fed HFC diet and orally treated with L-arginine or L-citrulline had higher relative percentage of 18:0, 20:0 and 22:6 and lower 16:0 fatty acids than rats fed HFC diet. Light and transmission electron microscopic findings of the thoracic aorta confirmed the biochemical results and demonstrated structural changes in the endothelial cells of the intimal layer, medial smooth muscle cells as well as in the adventitial layer in HFC fed-animals. However, these findings indicate little structural alterations in animals supplemented with L-arginine or L-citrulline along with HFC feeding. In the present study, L-arginine or L-citrulline was effective hypocholesterolemic and hypolipidemic agents in rats.