An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Favourable long-term outcome after immediate treatment of neonatal hyperammonemia due to N-acetylglutamate synthase deficiency

INTRODUCTION: N-Acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder, which may present in the neonatal period with severe hyperammonemia and marked neurological impairment. CASE REPORT: We report on a Turkish family with a patient who died due to hyperammonemia in the neonatal period. Reduced activity of NAGS and carbamyl phosphate synthetase were found at autopsy. A second child who developed hyperammonemia on the second day of life was immediately treated with arginine hydrochloride, sodium benzoate and protein restriction. After NAGS deficiency was suspected by enzyme analysis, sodium benzoate was replaced by N-carbamylglutamate (NCG). A third child who developed slight hyperammonemia on the third day of life was treated with NCG before enzyme analysis confirmed reduced NAGS activity. Neither of the patients developed hyperammonemia in the following years. After the human NAGS gene was identified, mutation analysis revealed that the older sibling on NCG therapy was homozygous for a 971G>A (W324X) mutation. The parents and the younger sibling were heterozygous. Therapy was continued in the older sibling until now without any adverse effects and favourable neurodevelopment outcome. In the younger sibling, therapy was stopped without any deterioration of urea cycle function. CONCLUSION: NAGS deficiency can be successfully treated with NCG and arginine hydrochloride with favourable outcome. Molecular diagnostic rather than enzyme analysis should be used in patients with suspected NAGS deficiency.

'Pergularain e I'--a plant cysteine protease with thrombin-like activity from Pergularia extensa latex

Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing approximately 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependent and time-dependent manner. At 10 microg concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues.

Role of selective V2-receptor-antagonism in septic shock: a randomized, controlled, experimental study

ABSTRACT : INTRODUCTION : V2-receptor (V2R) stimulation potentially aggravates sepsis-induced vasodilation, fluid accumulation and microvascular thrombosis. Therefore, the present study was performed to determine the effects of a first-line therapy with the selective V2R-antagonist (Propionyl1-D-Tyr(Et)2-Val4-Abu6-Arg8,9)-Vasopressin on cardiopulmonary hemodynamics and organ function vs. the mixed V1aR/V2R-agonist arginine vasopressin (AVP) or placebo in an established ovine model of septic shock. METHODS : After the onset of septic shock, chronically instrumented sheep were randomly assigned to receive first-line treatment with the selective V2R-antagonist (1 g/kg per hour), AVP (0.05 g/kg per hour), or normal saline (placebo, each n = 7). In all groups, open-label norepinephrine was additionally titrated up to 1 g/kg per minute to maintain mean arterial pressure at 70 ± 5 mmHg, if necessary. RESULTS : Compared to AVP- and placebo-treated animals, the selective V2R-antagonist stabilized cardiopulmonary hemodynamics (mean arterial and pulmonary artery pressure, cardiac index) as effectively and increased intravascular volume as suggested by higher cardiac filling pressures. Furthermore, left ventricular stroke work index was higher in the V2R-antagonist group than in the AVP group. Notably, metabolic (pH, base excess, lactate concentrations), liver (transaminases, bilirubin) and renal (creatinine and blood urea nitrogen plasma levels, urinary output, creatinine clearance) dysfunctions were attenuated by the V2R-antagonist when compared with AVP and placebo. The onset of septic shock was associated with an increase in AVP plasma levels as compared to baseline in all groups. Whereas AVP plasma levels remained constant in the placebo group, infusion of AVP increased AVP plasma levels up to 149 ± 21 pg/mL. Notably, treatment with the selective V2R-antagonist led to a significant decrease of AVP plasma levels as compared to shock time (P < 0.001) and to both other groups (P < 0.05 vs. placebo; P < 0.001 vs. AVP). Immunohistochemical analyses of lung tissue revealed higher hemeoxygenase-1 (vs. placebo) and lower 3-nitrotyrosine concentrations (vs. AVP) in the V2R-antagonist group. In addition, the selective V2R-antagonist slightly prolonged survival (14 ± 1 hour) when compared to AVP (11 ± 1 hour, P = 0.007) and placebo (11 ± 1 hour, P = 0.025). CONCLUSIONS : Selective V2R-antagonism may represent an innovative therapeutic approach to attenuate multiple organ dysfunction in early septic shock.

Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis

The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine-specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified.

Induction of p38, tumour necrosis factor-α and RANTES by mechanical stretching of keratinocytes expressing mutant keratin 10R156H

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma), characterized by ichthyotic, rippled hyperkeratosis, erythroderma and skin blistering, is a rare autosomal dominant disease caused by mutations in keratin 1 or keratin 10 (K10) genes. A severe phenotype is caused by a missense mutation in a highly conserved arginine residue at position 156 (R156) in K10.

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the L-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and L-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)- and heparin (KRSR)-binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Staphylococcus rostri sp. nov., a haemolytic bacterium isolated from the noses of healthy pigs

Twenty coagulase-negative Staphylococcus strains displaying alpha-haemolysis (delta-haemolysin) on sheep-blood agar were isolated from the noses of different pigs in Switzerland. The strains were Gram-stain-positive, non-motile cocci, catalase-positive and coagulase-negative. Sequence analysis of the 16S rRNA gene, sodA, rpoB, dnaJ and hsp60 and phylogenetic characteristics revealed that the strains showed the closest relatedness to Staphylococcus microti CCM 4903(T) and Staphylococcus muscae DSM 7068(T). The strains can be differentiated from S. microti by the absence of mannose fermentation and arginine arylamidase and from S. muscae by the absence of beta-glucuronidase activity and production of alkaline phosphatase. The chosen type strain ARI 262(T) shared 20.1 and 31.9 % DNA relatedness with S. microti DSM 22147(T) and S. muscae CCM 4903(T), respectively, by DNA-DNA hybridization. iso-C(15 : 0), anteiso-C(15 : 0) and iso-C(17 : 0) were the most common fatty acids. Cell-wall structure analysis revealed the peptidoglycan type A3alpha l-Lys-Gly(2)-l-Ser-Gly (type A11.3). The presence of teichoic acid was determined by sequencing the N-acetyl-beta-d-mannosaminyltransferase gene tarA, which is involved in biosynthesis of ribitol teichoic acid. Menaquinone 7 (MK-7) was the predominant respiratory quinone. The G+C content of ARI 262(T) was 38.8 mol%. The isolated strains represent a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus rostri sp. nov. The type strain is ARI 262(T) (=DSM 21968(T) =CCUG 57266(T)) and strain ARI 602 (=DSM 21969 =CCUG 57267) is a reference strain.

Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.

Dog bites man or man bites dog? The enigma of the amino acid conjugations

The proposition posed is that the value of amino acid conjugation to the organism is not, as in the traditional view, to use amino acids for the detoxication of aromatic acids. Rather, the converse is more likely, to use aromatic acids that originate from the diet and gut microbiota to assist in the regulation of body stores of amino acids, such as glycine, glutamate, and, in certain invertebrates, arginine, that are key neurotransmitters in the central nervous system (CNS). As such, the amino acid conjugations are not so much detoxication reactions, rather they are homeostatic and neuroregulatory processes. Experimental data have been culled in support of this hypothesis from a broad range of scientific and clinical literature. Such data include the low detoxication value of amino acid conjugations and the Janus nature of certain amino acids that are both neurotransmitters and apparent conjugating agents. Amino acid scavenging mechanisms in blood deplete brain amino acids. Amino acids glutamate and glycine when trafficked from brain are metabolized to conjugates of aromatic acids in hepatic mitochondria and then irreversibly excreted into urine. This process is used clinically to deplete excess nitrogen in cases of urea cycle enzymopathies through excretion of glycine or glutamine as their aromatic acid conjugates. Untoward effects of high-dose phenylacetic acid surround CNS toxicity. There appears to be a relationship between extent of glycine scavenging by benzoic acid and psychomotor function. Glycine and glutamine scavenging by conjugation with aromatic acids may have important psychosomatic consequences that link diet to health, wellbeing, and disease.

Vasopressin in vasodilatory shock: ensure organ blood flow, but take care of the heart!

Supplementary arginine vasopressin infusion in advanced vasodilatory shock may be accompanied by a decrease in cardiac index and systemic oxygen transport capacity in approximately 40% of patients. While a reduction of cardiac output most frequently occurs in patients with hyperdynamic circulation, it is less often observed in patients with low cardiac index. Infusion of inotropes, such as dobutamine, may be an effective strategy to restore systemic blood flow. However, when administering inotropic drugs, systemic blood flow should be increased to adequately meet systemic demands (assessed by central or mixed venous oxygen saturation) without putting an excessive beta-adrenergic stress on the heart. Overcorrection of cardiac index to hyperdynamic values with inotropes places myocardial oxygen supply at significant risk.

An unusual patient with hypercalciuria, recurrent nephrolithiasis, hypomagnesemia and G227R mutation of Paracellin-1. An unusual patient with hypercalciuria and hypomagnesemia unresponsive to thiazide diuretics

A 19-year-old female patient with hypercalciuria and recurrent nephrolithiasis/urinary tract infection unresponsive to thiazide type diuretics is presented. The patient first experienced nephrolithiasis at the age of 4 years. Afterwards, recurrent passages of stones and urinary tract infection occurred. On diagnostic evaluation at the age of 19 years, she also had hypocitraturia and hypomagnesemia. Her serum calcium concentrations were near the lower limit of normal (8.5-8.8 mg/dl; normal range: 8.5-10.5), her serum magnesium concentrations were 1.15-1.24 mg/dl (normal range: 1.4-2.5) and urinary calcium excretion was 900 mg/24 h. PTH concentrations were increased (110-156 pg/ml; normal range: 10-65). We tried to treat the patient with hydrochlorothiazide at a dose of 50 mg/day. During treatment with thiazide diuretics, PTH concentration remained high and the patient had recurrent urinary tract infections and passages of stones. Serum magnesium concentration did not normalize even under the parenteral magnesium infusion. Her mother had a history of nephrolithiasis 20 years ago. Severe hypomagnesemia in association with hypercalciuria/urinary stones is reported as a rare autosomal recessive disorder caused by impaired reabsorption of magnesium and calcium in the thick assending limp of Henle's loop. Recent studies showed that mutations in the CLDN16 gene encoding paracellin-1 cause the disorder. In exon 4, a homozygous nucleotide exchange (G679C) was identified for the patient. This results in a point mutation at position Glycine227, which is replaced by an Arginine residue (G227R). The mother was heterozygous for this mutation. G227 is located in the fourth transmembrane domain and is highly conserved in the claudin gene family. This case indicates the pathogenetic role of paracellin-1 mutation in familial hypomagnesemia with hypercalciuria and nephrocalcinosis and further underlines the risk of stone formation in heterozygous mutation carriers.

Absence of histamine-induced nitric oxide release in the human radial artery: implications for vasospasm of coronary artery bypass vessels

Radial artery (RA) bypass grafts can develop severe vasospasm. As histamine is known to induce vasospasm, its effect on RA was assessed compared with the classic bypass vessels internal mammary artery (MA) and saphenous vein (SV). The vessels were examined in organ chambers for isometric tension recording. Histamine induced contractions on baseline; the sensitivity was higher in RA and SV than MA. After precontraction with norepinephrine, histamine did not evoke relaxations of RA but induced relaxations of MA and less of SV at lower concentrations; it induced contractions at higher concentrations, reaching similar levels in all three vessels. Indomethacin did not affect the response of MA and RA but potentiated relaxations and reduced contractions of SV. Endothelium removal, N(omega)-nitro-L-arginine methyl ester (L-NAME), or the H2-receptor blocker cimetidine did not affect the response of RA, but inhibited relaxations and enhanced contractions in MA and inhibited relaxations in SV; in the latter, only L-NAME enhanced contractions. Real-time PCR detected much lower expression of endothelial H2-receptor in RA than MA or SV. Western blots revealed similar endothelial nitric oxide (NO) synthase expression in all three vessels. Relaxations to acetylcholine were identical in RA and MA. Thus histamine releases NO by activating the endothelial H2-receptor, the expression of which is much lower in RA than MA or SV. H2-receptor activation also releases prostaglandins in SV, partially antagonizing NO. The lack of histamine-induced NO production represents a possible mechanism of RA vasospasm.