Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potential in biotechnology and medicine. We prepared several histidine analogues including a novel histidine analogue, beta-(1,2,3-triazol-4-yl)-DL-alanine. These histidine analogues were assayed for translational activity in histidine-auxotrophic Escherichia coli strain UTH780. We observed that several histidine analogues, including our novel histidine analogue, were efficiently incorporated into the protein in vivo; however, other analogues were rejected. These results suggest that the hydrogen atom at a specific position seriously affects incorporation.     

Simvastatin prevents oxygen and glucose deprivation/reoxygenation-induced death of cortical neurons by reducing the production and toxicity of 4-hydroxy-2E-nonenal

Lipid membrane peroxidation is highly associated with neuronal death in various neurodegenerative diseases including cerebral stroke. Here, we report that simvastatin decreases oxygen and glucose deprivation (OGD)/reoxygenation-evoked neuronal death by inhibiting the production and cytoxicity of 4-hydroxy-2E-nonenal (HNE), the final product of lipid peroxidation. Simvastatin markedly decreased the OGD/reoxygenation-evoked death of cortical neurons. OGD/reoxygenation increased the intracellular HNE level mostly in neuronal cells, not glial cells. Simvastatin decreased the intracellular level of HNE in neuronal cells exposed to OGD/reoxygenation. We further found that HNE induced the cytotoxicity in neuronal cells and synergistically increased the N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity. Simvastatin largely blocked the NMDA neurotoxicity potentiated by HNE. However, simvastatin did not alter the NMDA-evoked calcium influx in the absence or presence of HNE. HNE inhibited the activity of nuclear factor-kappa B (NF-kappaB), and the cytotoxicity of HNE was in good correlation with inactivation of NF-kappaB. Simvastatin reversed the inhibition of NF-kappaB activity induced by OGD/reoxygenation or HNE. The neuroprotection by simvastatin was significantly attenuated by various NF-kappaB inhibitors, implying that simvastatin inhibits the cytotoxicity of HNE at least in part by maintaining the activity of NF-kappaB. Further understanding of the neuroprotective mechanism of simvastatin may provide a therapeutic strategy for oxidative stress-related neurodegenerative diseases.     

Two-dimensional NMR studies of the zinc finger motif: solution structures and dynamics of mutant ZFY domains containing aromatic substitutions in the hydrophobic core.

Solution structures of mutant Zn fingers containing aromatic substitutions in the hydrophobic core are determined by 2D-NMR spectroscopy and distance-geometry/simulated annealing (DG/SA). The wild-type domain (designated ZFY-6) is derived from the human male-associated protein ZFY and represents a sequence motif (Cys-X2-Cys-X-Ar-X7-Leu-X2-His-X4-His) that differs from the consensus (Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-His-X3-His) in the location ("aromatic swap") and diversity (Ar = tyrosine, phenylalanine, or histidine) of the central aromatic residue (underlined). In a given ZFY domain the choice of a particular aromatic residue is invariant among vertebrates, suggesting that alternative "swapped" aromatic residues are functionally inequivalent. 2D-NMR studies of analogues containing tyrosine, phenylalanine, or histidine at the swapped site yield the following results. (i) The three DG/SA structures each retain the beta beta alpha motif and exhibit similar staggered-horizontal packing between the variant aromatic residue and the proximal histidine in the hydrophobic core. (ii) The structures and stabilities of the tyrosine and phenylalanine analogues are essentially identical, differing only by local exposure of polar (Tyr p-OH) or nonpolar (Phe p-H) surfaces. (iii) The dynamic stability of the histidine analogue is reduced as indicated by more rapid protein-deuterium exchange of hydrogen bonds related to secondary structure and amide-sulfur coordination (slowly exchanging amide resonances in D2O) and by more extensive averaging of main-chain dihedral angles (3J alpha NH coupling constants). An aspartic acid in the putative DNA recognition surface, whose configuration is well-defined as a possible helix N-cap in the tyrosine and phenylalanine analogues, exhibits multiple weak main-chain contacts in the NOESY spectrum of the histidine analogue; such NOEs are geometrically inconsistent and so provide complementary evidence for structural fluctuations. (iv) Because the three DG ensembles have similar apparent precision, the finding of reduced dynamic stability in the histidine analogue emphasizes the importance of experiments that directly probe fluctuations at several time scales. Our results provide insight into the design of biological metal-binding sites and the relationship of protein sequence to structure and dynamics.     

Pretreatment with 3-O-methyl-d-glucose or 2-deoxy-d-glucose attenuates the post-mortem rise in rat brain lactate

The present investigation examined the effects of pretreatment with 3-O-methyl-d-glucose (3OMG) or 2-deoxy-d-glucose (2DOG) on post-mortem rise in rat brain lactate to evaluate their potential use for minimizing ischemia-induced rise in brain lactate. The results showed that iv administration of either glucose analogue (2 g/kg) at 2.5 min prior to sacrifice significantly attenuated (to 0.61 of control levels) post-mortem brain lactate rise. Pretreating rats with 2-deoxy-d-glucose (2 g/kg) 15 min prior to sacrifice resulted in a greater inhibition (to 0.52 of control) of the post-mortem lactate rise. The effects of these two analogues (3OMG and 2DOG) can be accounted for by their inhibition of brain glucose transport and inhibition of brain glucose metabolism by 2DOG. The present results suggest that intervention with either of these glucose analogues under the proper experimental procedures may minimize the cytopathological consequences of ischemia related to the rise in brain lactate.     

Stimulation of protein synthesis by lysine analogues in lysine-deprived Ehrlich ascites tumour cells

This paper describes experiments in which we have investigated the mechanism by which amino acid starvation regulates the initiation of protein synthesis in mammalian cells. We have examined the ability of a range of lysine analogues to stimulate protein synthesis in lysine-deprived mouse Ehrlich ascites tumour cells in culture. Of those analogues tested, only those which are cleaved to lysine intracellularly are capable of restoring protein synthesis to the level seen in fully fed cells. Lysine which is covalently linked to agarose does not stimulate translation. After 5 min incubation of lysine-deprived cells with the analogue lysine p-nitroanilide, the lysine concentration in cell extracts is restored to that found in extracts from fed cells, and protein synthesis is maximally stimulated within 5–10 min. During this period of time there is no increase in the concentration of lysine in the medium. These data indicate that it is the size of the intracellular rather than the extracellular amino acid pool which regulates the rate of protein synthesis during amino acid deprivation.     

Complement component 3 inhibition by an antioxidant is neuroprotective after cerebral ischemia and reperfusion in mice

Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that pro‐inflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up‐regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen‐glucose deprivation in brain cells. Persistent up‐regulation of complement component 3 expression was reduced in copper/zinc‐superoxide dismutase transgenic mice, and manganese‐superoxide dismutase knock‐out mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N‐tert‐butyl‐α‐phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N‐tert‐butyl‐α‐phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen‐glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke.     

Administration of D-glucosamine into the third cerebroventricle induced feeding accompanied by hyperglycemia in rats

D-glucosamine, 2-amino-2-deoxy-D-glucose, is known to be an endogenous glucose analogue and to antagonize glucose uptake and metabolism. The present experiments were aimed to clarify effects of glucosamine and related chemical substances on ingestive behavior, as well as its direct effects on hypothalamic neurons. Infusion of 24 mumole glucosamine into the third cerebroventricle induced feeding within 30 min in 5 rats out of 7 tested, accompanied by increased ambulatory activity. No periprandial drinking was observed. Plasma glucose level increased, peaking at 30 min after the injection. Plasma insulin level tended to increase, but not significantly. Electrophoretic application of glucosamine activated glucose-sensitive neurons in the lateral hypothalamus and suppressed glucoreceptors in the ventromedial hypothalamus. These facts, together with other reported results, suggest that glucosamine can modulate physiological feeding and that carbon 2 of the glucose molecule is important in feeding modulation by glucose analogues.     

Nondestructive Measurement of Retinal Glucose Transport and Consumption In Vivo Using NMR Spectroscopy

Abstract: The cellular events underlying various retinopathies are poorly understood but likely involve perturbation of retinal glucose metabolism. Current methods for assessing this metabolism are destructive, thus limiting longitudinal studies. We hypothesize that following an intravitreous injection, the clearance rate of a glucose analogue will be a nondestructive index of retinal glucose transport and metabolism in vivo. First, radiolabeled glucose analogues were injected into the vitreous. After 40 min, the dominant clearance path was posterior via the retina and was consistent with a facilitated transport mechanism. Next, either [6,6-²H₂]glucose or 3-deoxy-3-fluoro- d -glucose was injected into the vitreous of rabbit eyes, and the clearance rate of each analogue was determined over 40 min using, respectively, ²H or ¹⁹F NMR. These rates were interpreted as a function of the retinal glucose transport and consumption. From the NMR data, the rate of retinal glucose consumption was ∼16 times slower than the transport of glucose. These data demonstrate that NMR measurements of glucose analogue clearance rate from the vitreous can provide a nondestructive index of retinal glucose transport and consumption in vivo.     

Induced Structural Defects in T-Even Bacteriophage

Multiple aberrant substructures of T-even bacteriophage particles occurred when amino acid analogues or antimetabolites were present during phage growth. Certain aberrant substructures were induced by specific analogues or antimetabolites. In particular, it was observed by electron microscopy that l-canavanine, an arginine analogue, gave rise to polyheads; l-azetidine-2-carboxylic acid, a proline analogue, gave rise to polytail tubes; and 1,2,4-trizaole-3-alanine, a histidine analogue, proflavine, and actinomycin D all gave rise to small heads. These aberrant substructures were similar to those reported earlier with conditional lethal mutants (amber) of T4D in a restrictive host.     

Monoclonal Antibodies for Detection of 4-Hydroxynonenal Modified Proteins

A promising approach to study lipid peroxidation pathology is antibodies recognizing aldehydes which react with and became bound to amino acid side chains of proteins. We present in this study the characterization of several monoclonal antibodies which recognize 4-hydroxynonenal (HNE) modified proteins. Six out of 20 antibodies recognizing HNE modified BSA were able to detect HNE-protein adducts in peroxidized liver microsomes. Two of these antibodies were selected and characterized. Both antibodies could also detect HNE-protein adducts in oxidized low density lipoprotein. They exhibit no detectable cross reaction with proteins modified by malonaldehyde, nonanal, nonenal and 4-hydroxyhexenal. Protein bound 4-hydroxyoctenal and 4-hydroxydecenal were recognized to some extent. Further characterization revealed that the two antibodies are highly selective for HNE bound to histidine with only some cross reaction to HNE bound to lysine and cysteine. Preliminary quantitative ELISA-analysis showed that oxidized microsomes and oxidized LDL contain 12 nmol and 3 nmol HNE-histidine per mg protein respectively.     

Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.     

Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal

The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (1 mM) at 4, 25, and 37 degrees C at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degrees C, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degrees C, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T(m) for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.     

The A14 position of insulin tolerates considerable structural alterations with modest effects on the biological behavior of the hormone

As part of our aim to investigate the contribution of the tyrosine residue found in the 14 position of the A-chain to the biological activity of insulin, we have synthesized six insulin analogues in which the A14 Tyr has been substituted by a variety of amino acid residues. We have selected three hydrophilic and charged residues—glutamic acid, histidine, and lysine—as well as three hydrophobic residues—cycloleucine, cyclohexylalanine, and naphthyl-(1)-alanine—to replace the A14 Tyr. All six analogues exhibit full agonist activity, reaching the same maximum stimulation of lipogenesis as is achieved with procine insulin. The potency for five of the six analogues, [A14 Glu]-, [A14 His]-, [A14 Lys]-, [A14 cycloleucine]-, and [A14 naphthyl-(1)-alanine]-insulins in receptor binding assays ranges from 40–71% and in stimulation of lipogenesis ranges from 35-120% relative to porcine insulin. In contrast, the potency of the sixth analogue, [A14 cyclohexylalanine]insulin, in both types of assays is less than 1% of the natural hormone. The retention time on reversed-phase high-performance liquid chromatography for the first five analogues is similar to that of bovine insulin, whereas for the sixth analogue, [A14 cyclohexylalanine]insulin, it is approximately 11 min longer than that of the natural hormone. This suggests a profound change in conformation of the latter analogue. Apparently, the A14 position of insulin can tolerate a wide latitude of structural alterations without substantial decrease in potency. This suggests that the A14 position does not participate directly in insulin receptor interaction. Only when a substitution which has the potential to disrupt the conformation of the molecule is made at this position, is the affinity for the receptor, and hence the biological potency, greatly reduced.     

The Lipid Peroxidation Product 4-Hydroxynonenal Inhibits Neurite Outgrowth, Disrupts Neuronal Microtubules, and Modifies Cellular Tubulin

Oxidative stress is believed to be an important factor in the development of age-related neurodegenerative diseases such as Alzheimer's disease (AD). The CNS is enriched in polyunsaturated fatty acids and is therefore particularly vulnerable to lipid peroxidation. Indeed, accumulation of lipid peroxidation products has been demonstrated in affected regions in brains of AD patients. Another feature of AD is a change in neuronal microtubule organization. A possible causal relationship between lipid peroxidation products and changes in neuronal cell motility and cytoskeleton has not been investigated. We show here that 4-hydroxy-2(E)-nonenal (HNE), a major product of lipid peroxidation, inhibits neurite outgrowth and disrupts microtubules in Neuro 2A cells. The effect of HNE on microtubules was rapid, being observed after incubation times as short as 15 min. HNE can react with target proteins by forming either Michael adducts or pyrrole adducts. 4-Oxononanal, an HNE analogue that can form only pyrrole adducts but not Michael adducts, had no effect on the microtubules. This suggests that the HNE-induced disruption of microtubules occurs via Michael addition. We also show that cellular tubulin is one of the major proteins modified by HNE and that the HNE adduction to tubulin occurs via Michael addition. Inhibition of neurite outgrowth, disruption of microtubules, and tubulin modification were observed at pathologically relevant HNE concentrations and were not accompanied by cytotoxicity. Our results show that these are proximal effects of HNE that may contribute to cytoskeletal alterations that occur in AD.     

Glucose as a regulator of insulin-sensitive hexose uptake in 3T3 adipocytes

In the present study we examined the role of glucose in the regulation of its own transport activity in the cultured 3T3 fat cell. A regulatory control of glucose became apparent after these cells were cultured in the absence of glucose. Glucose deprivation of the cells was accompanied by a specific time and protein synthesis-dependent increase in dGlc (2-deoxyglucose) uptake (up to 5-fold), which was due to an increase in the apparent Vmax of the transport system. Concomitantly, the stimulatory effect of insulin on hexose uptake almost completely disappeared. Addition of glucose to the glucose-deprived cells rapidly reversed the deprivation effects. Cycloheximide experiments revealed that the glucose deprivation-induced increase in hexose uptake required protein synthesis as well as a protein synthesis-independent response to glucose deprivation that retarded the turnover of hexose transport activity. Taken together, these data indicate that glucose deprivation is accompanied by retardation of the rate of degradation, internalization, or inactivation of hexose transporters while the increase in dGlc uptake requires at least the continuation of protein synthesis-dependent de novo synthesis, insertion, or activation of hexose transporters. Hexose competitively taken up with dGlc, including the nonmetabolizable glucose analogue 3-O-methylglucose, could replace glucose in the process of prevention and reversal of the deprivation effects, indicating that competitive transport but not the metabolism of hexose is a prerequisite for the regulatory effect of glucose on the activity of its own transport system. In conclusion, our results indicate that in cultured 3T3 fat cells glucose itself is involved in the regulation of the activity of its own transport system by influencing the rate of degradation, internalization, or inactivation of hexose transporters by a protein synthesis-independent mechanism.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Interleukin-6 protects PC12 cells from 4-hydroxynonenal-induced cytotoxicity by increasing intracellular glutathione levels

Oxidative stress plays an important role in neuronal cell death associated with many different neurodegenerative conditions, and it is reported that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal cell death induced by oxidative stress. Previously, we have demonstrated that interleukin-6 (IL-6) protects PC12 cells from serum deprivation and 6-hydroxydopamine-induced toxicity. Therefore, in the present study, we examined the effects of interleukins on HNE toxicity in PC12 cells. Exposure of PC12 cells to HNE resulted in a decrease in levels of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, which was due to necrotic and apoptotic cell death. Addition of IL-6 24 h before HNE treatment provided a concentration-dependent protection against HNE toxicity, whereas neither IL-1β nor IL-2 had any effect. Addition of glutathione (GSH)-ethyl ester, but not superoxide dismutase or catalase, before HNE treatment to the culture medium protected PC12 cells from HNE toxicity. We found that IL-6 increases intracellular GSH levels and the activity of γ-glutamylcysteine synthetase (γ-GCS) in PC12 cells. Buthionine sulfoximine (BSO), an inhibitor of γ-GCS, reversed the protective effect of IL-6 against HNE toxicity. These results suggest that IL-6 protects PC12 cells from HNE-induced cytotoxicity by increasing intracellular levels of GSH.     

The interaction of amiloride analogues with the Na+/H+ exchanger in kidney medulla microsomes

The effects of ten amiloride analogues on Na+-H+ exchange in rabbit kidney medulla microsomes have been examined. Most of the analogues appeared to inhibit Na+ uptake into the microsomes more effectively than did amiloride either in the presence or absence of a pH gradient. However, the analogues were also capable of stimulating Na+ efflux from the microsomes at concentrations somewhat higher than the concentrations at which they inhibited Na+ influx. The concentrations at which the analogues stimulated Na+ efflux were about 2-4-times higher than the concentrations at which they blocked influx. This suggested that the two processes were related. The analogues that stimulated efflux most effectively (the 5-N-benzyl-amino analogue of amiloride and the 5-N-butyl-N-methylamino analogue) were shown to induce completely reversible effects. These analogues did not stimulate L-[3H]glucose efflux from medulla microsomes which ruled out nonspecific vesicle destruction or reversible detergent effects. These analogues also induced Na+ efflux from microsomes in the presence of high concentrations of added buffer, which ruled out weak-base uncoupling effects. The possibility exists that these analogues are carried into the microsomes via the Na+-H+ exchange protein and that this permits them to both block Na+ influx into the microsomes and stimulate Na+ efflux as well.     

Superactive insulins

The substitution of aspartic acid for the naturally-occurring histidine residue in position B10 in human insulin results in an insulin analogue which displays an in vitro potency 4- to 5-fold greater than the parent compound. This substitution has been introduced into six insulin analogues which, before modification, display potencies ranging from less than 0.01-fold to 3-fold relative to natural insulin. In each case, the resulting aspartic acid-substituted analogue is substantially more potent than the parent compound. Thus, it is now possible to prepare "tailor-made" insulins with enhanced potency.     

Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 approximately K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pK(a) values suggest that location in the hydrophobic binding cavity and low predicted pK(a) of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress.