Allylation of intraerythrocytic hemoglobin by raw garlic extracts.

Recent studies have shown that deoxygenated human red blood cells (RBCs) converted garlic-derived polysulfides into hydrogen sulfide, which in turn produced vasorelaxation in aortic ring preparations. The vasoactivity was proposed to occur via glucose- and thiol-dependent acellular reactions. In the present study, we investigated the interaction of garlic extracts with human deoxygenated RBCs and its effect on intracellular hemoglobin molecules. The results showed that garlic extract covalently modified intraerythrocytic deoxygenated hemoglobin. The modification identified consisted of an addition of 71 atomic mass units, suggesting allylation of the cysteine residues. Consistently, purified human deoxyhemoglobin reacted with chemically pure diallyl disulfide, showing the same modification as garlic extracts. Tandem mass spectrometry analysis demonstrated that garlic extract and diallyl disulfide modified hemoglobin's beta-chain at cysteine-93 (beta-93C) or cysteine-112 (beta-112C). These results indicate that garlic-derived organic disulfides as well as pure diallyl disulfide must permeate the RBC membrane and modified deoxyhemoglobin at beta-93C or beta-112C. Although the physiological role of the reported garlic extract-induced allyl modification on human hemoglobin warrants further study, the results indicate that constituents of natural products, such as those from garlic extract, modify intracellular proteins.

Noninvasive monitoring of tissue hemoglobin using UV-VIS diffuse reflectance spectroscopy: a pilot study.

We conducted a pilot study on 10 patients undergoing general surgery to test the feasibility of diffuse reflectance spectroscopy in the visible wavelength range as a noninvasive monitoring tool for blood loss during surgery. Ratios of raw diffuse reflectance at wavelength pairs were tested as a first-pass for estimating hemoglobin concentration. Ratios can be calculated easily and rapidly with limited post-processing, and so this can be considered a near real-time monitoring device. We found the best hemoglobin correlations were when ratios at isosbestic points of oxy- and deoxyhemoglobin were used, specifically 529/500 nm. Baseline subtraction improved correlations, specifically at 520/509 nm. These results demonstrate proof-of-concept for the ability of this noninvasive device to monitor hemoglobin concentration changes due to surgical blood loss. The 529/500 nm ratio also appears to account for variations in probe pressure, as determined from measurements on two volunteers.

Rapid ratiometric determination of hemoglobin concentration using UV-VIS diffuse reflectance at isosbestic wavelengths.

We developed a ratiometric method capable of estimating total hemoglobin concentration from optically measured diffuse reflectance spectra. The three isosbestic wavelength ratio pairs that best correlated to total hemoglobin concentration independent of saturation and scattering were 545/390, 452/390, and 529/390 nm. These wavelength pairs were selected using forward Monte Carlo simulations which were used to extract hemoglobin concentration from experimental phantom measurements. Linear regression coefficients from the simulated data were directly applied to the phantom data, by calibrating for instrument throughput using a single phantom. Phantoms with variable scattering and hemoglobin saturation were tested with two different instruments, and the average percent errors between the expected and ratiometrically-extracted hemoglobin concentration were as low as 6.3%. A correlation of r = 0.88 between hemoglobin concentration extracted using the 529/390 nm isosbestic ratio and a scalable inverse Monte Carlo model was achieved for in vivo dysplastic cervical measurements (hemoglobin concentrations have been shown to be diagnostic for the detection of cervical pre-cancer by our group). These results indicate that use of such a simple ratiometric method has the potential to be used in clinical applications where tissue hemoglobin concentrations need to be rapidly quantified in vivo.

Androesterilidad genético citoplasmática en sorgo : empleo de los citoplasmas A2 yA3

p.117-122

Ca2+-independent phospholipase A2-dependent gating of TRPM8 by lysophospholipids

TRPM8 represents an ion channel activated by cold temperatures and cooling agents, such as menthol, that underlies the cold-induced excitation of sensory neurons. Interestingly, the only human tissue outside the peripheral nervous system, in which the expression of TRPM8 transcripts has been detected at high levels, is the prostate, a tissue not exposed to any essential temperature variations. Here we show that the TRPM8 cloned from human prostate and heterologously expressed in HEK-293 cells is regulated by the Ca(2+)-independent phospholipase A(2) (iPLA(2)) signaling pathway with its end products, lysophospholipids (LPLs), acting as its endogenous ligands. LPLs induce prominent prolongation of TRPM8 channel openings that are hardly detectable with other stimuli (e.g. cold, menthol, and depolarization) and that account for more than 90% of the total channel open time. Down-regulation of iPLA(2) resulted in a strong inhibition of TRPM8-mediated functional responses and abolished channel activation. The action of LPLs on TRPM8 channels involved either changes in the local lipid bilayer tension or interaction with the critical determinant(s) in the transmembrane channel core. Based on this, we propose a novel concept of TRPM8 regulation with the involvement of iPLA(2) stimulation. This mechanism employs chemical rather than physical (temperature change) signaling and thus may be the main regulator of TRPM8 activation in organs not exposed to any essential temperature variations, as in the prostate gland.

Synthesis of (+)-piclavines A1 and A2

Piclavines AI and A2 have been synthesised for the first time. The route is short with the key step being the reaction of a bicyclic N-acyl iminium ion with 3-trimethysilyl-1-decene. This convergent strategy gave exclusively compounds in which the pendant decenyl group was axial, as a 6:1 mixture of E:Z-alkene diastereoisomers. Reduction of the lactam carbonyl group gave a 6:1 mixture of piclavines Al and A2, (C) 2000 Published by Elsevier Science Ltd.

Stroma-free hemoglobin increases blood pressure and GFR in the hypotensive rat: role of nitric oxide.

The short-term systemic and renal hemodynamic effects of two stroma-free hemoglobin (SFH) solutions, one unmodified and the other modified by cross-linking, were examined in anesthetized rats after hemorrhagic hypotension. Both forms of SFH increased mean arterial pressure (MAP) and glomerular filtration rate (GFR) to baseline (prehemorrhage) values. The increase in MAP induced by unmodified SFH was greater than the increase in MAP caused by an albumin solution isoncotic to the unmodified SFH solution. Similarly, the increase in MAP caused by the modified SFH was also substantially greater than that induced by an albumin solution of comparable oncotic pressure to the modified SFH solution. Both unmodified and modified SFH increased GFR. As with MAP, the increase in GFR induced by both SFH solutions was greater than that associated with the oncotically matched albumin solutions. In separate experiments, the effects of nitric oxide (NO) inhibition with N omega-nitro-L-arginine methyl ester (L-NAME) on MAP after hemorrhagic hypotension and subsequent infusion of unmodified SFH or albumin were also examined. In the albumin-infused rats, L-NAME increased MAP. In marked contrast, NO inhibition with L-NAME had no further effect on MAP when infused after SFH. We conclude that both unmodified and modified SFH solutions acutely improve MAP and GFR by the combined effects of intravascular volume expansion resulting from the colloid effect of the protein and by inactivation of NO.

Posttranscriptional regulation of acute phase serum amyloid A2 expression by the 5'- and 3'-untranslated regions of its mRNA

Human acute-phase serum amyloid A protein (A-SAA) is a major acute phase reactant, the concentration of which increases dramatically as part of the body's early response to inflammation. A-SAA is the product of two almost identical genes, SAA1 and SAA2, which are induced by the pro-inflammatory cytokines, IL-1 and IL-6. In this study, we examine the roles played by the 5'- and 3'-untranslated regions (UTRs) of the SAA2 mRNA in regulating A-SAA2 expression. SAA2 promoter-driven luciferase reporter gene constructs carrying the SAA2 5'-UTR and/or 3'-UTR were transiently transfected into the HepG2 human hepatoma cell line. After induction of chimeric mRNA with IL-1beta and IL-6, the SAA2 5'- and 3'-UTRs were both able to posttranscriptionally modify the expression of the luciferase reporter. The SAA2 5'-UTR promotes efficient translation of the chimeric luciferase transcripts, whereas the SAA2 3'-UTR shares this property and also significantly accelerates the rate of reporter mRNA degradation. Our data strongly suggest that the SAA2 5'- and 3'-UTRs each play significant independent roles in the posttranscriptional regulation of A-SAA2 protein synthesis.

Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.

Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom--synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region

Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.