Use of the environmentally sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide to study peptoid helix structures

Peptoids, oligomers of N-substituted glycine, have been valuable targets for study and diverse application as peptidomimetics and as nanomaterials. Their conformational heterogeneity has made the study of peptoid structures using high-resolution analyses challenging, limiting our understanding of the physiochemical features that mediate peptoid folding. Here, we introduce a new method for the study of peptoid structure that relies on the environmentally sensitive fluorescence properties of 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN). We have prepared a 4-DMN-functionalized primary amine that is compatible with the traditional submonomer peptoid synthesis methods and incorporated it sequence-specifically into 11 of 13 new peptoids. When included as a peptoid side chain modification, the fluorescence emission intensity of 4-DMN correlates with predictions of the fluorophore's local polarity within a putative structure. 4-DMN fluorescence is maximized when the fluorophore is placed in the middle of the hydrophobic face of an amphiphilic helical peptoid. When the fluorophore is placed near the peptoid terminus or on a polar face of an amphiphilic sequence, 4-DMN fluorescence is diminished. Disruption of the peptoid secondary structure or amphiphilicity also modulates 4-DMN fluorescence. The peptoids' helical secondary structures are moderately disrupted by inclusion of a 4-DMN-modified side chain as evaluated by changes in the peptoids' CD spectral features. This new method for peptoid structure evaluation should be a valuable complement to existing peptoid structural analysis tools.     

Is sprint exercise a leptin signaling mimetic in human skeletal muscle?

This study was designed to determine whether sprint exercise activates signaling cascades linked to leptin actions in human skeletal muscle and how this pattern of activation may be interfered by glucose ingestion. Muscle biopsies were obtained in 15 young healthy men in response to a 30-s sprint exercise (Wingate test) randomly distributed into two groups: the fasting (n = 7, C) and the glucose group (n = 8, G), who ingested 75 g of glucose 1 h before the Wingate test. Exercise elicited different patterns of JAK2, STAT3, STAT5, ERK1/2, p38 MAPK phosphorylation, and SOCS3 protein expression during the recovery period after glucose ingestion. Thirty minutes after the control sprint, STAT3 and ERK1/2 phosphorylation levels were augmented (both, P < 0.05). SOCS3 protein expression was increased 120 min after the control sprint but PTP1B protein expression was unaffected. Thirty and 120 min after the control sprint, STAT5 phosphorylation was augmented (P < 0.05). Glucose abolished the 30 min STAT3 and ERK1/2 phosphorylation and the 120 min SOCS3 protein expression increase while retarding the STAT5 phosphorylation response to sprint. Activation of these signaling cascades occurred despite a reduction of circulating leptin concentration after the sprint. Basal JAK2 and p38 MAPK phosphorylation levels were reduced and increased (both P < 0.05), respectively, by glucose ingestion prior to exercise. During recovery, JAK2 phosphorylation was unchanged and p38 MAPK phosphorylation was transiently reduced when the exercise was preceded by glucose ingestion. In conclusion, sprint exercise performed under fasting conditions is a leptin signaling mimetic in human skeletal muscle.     

Hydrogen sulfide is a partially redox-independent activator of the human jejunum Na+ channel, Nav1.5

Hydrogen sulfide (H(2)S) is produced endogenously by L-cysteine metabolism. H(2)S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H(2)S donor NaHS on Na(V)1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H(2)S acts on Na(V)1.5 by redox reactions. Whole cell Na(+) currents were recorded in freshly dissociated human jejunum circular myocytes and Na(V)1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H(2)S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na(+) peak currents and shifted the half-point (V(1/2)) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed Na(V)1.5 α subunit with EC(50)s in the 10(-4) to 10(-3) M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg(2+)-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of Na(V)1.5 peak current. These studies show that H(2)S activates the gastrointestinal Na(+) channel, and the mechanism of action of H(2)S is partially redox independent.     

Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H(+) NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H(+) NK cells, may be not be as robust as the secondary response observed in T cells.     

Cross-resistance to insecticides in a malathion-resistant strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.     

Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation

There is a significant flux of the neurotoxic oxysterol 27-hydroxycholesterol (27OHC) from the circulation across the blood-brain barrier. Because there is a correlation between 27OHC and cholesterol in the circulation and lipoprotein-bound cholesterol does not pass the blood-brain barrier, we have suggested that 27OHC may mediate the effects of hypercholesterolemia on the brain. We previously demonstrated a modest accumulation of 27OHC in brains of patients with sporadic Alzheimer's disease (AD), consistent with a role of 27OHC as a primary pathogenetic factor. We show here that there is a 4-fold accumulation of 27OHC in different regions of the cortexes of patients carrying the Swedish amyloid precursor protein (APPswe) 670/671 mutation. The brain levels of sitosterol and campesterol were not significantly different in the AD patients compared with the controls, suggesting that the blood-brain barrier was intact in the AD patients. We conclude that accumulation of 27OHC is likely to be secondary to neurodegeneration, possibly a result of reduced activity of CYP7B1, the neuronal enzyme responsible for metabolism of 27OHC. We discuss the possibility of a vicious circle in the brains of the patients with familial AD whereby neurodegenerative changes cause an accumulation of 27OHC that further accelerates neurodegeneration.     

Second-site compensatory mutations of HIV-1 capsid mutations

The human immunodeficiency virus (HIV) capsid (CA) protein assembles into a hexameric lattice that forms the mature virus core. Contacts between the CA N-terminal domain (NTD) of one monomer and the C-terminal domain (CTD) of the adjacent monomer are important for the assembly of this core. In this study, we have examined the effects of mutations in the NTD region associated with this interaction. We have found that such mutations yielded modest reductions of virus release but major effects on viral infectivity. Cell culture and in vitro assays indicate that the infectivity defects relate to abnormalities in the viral cores. We have selected second-site compensatory mutations that partially restored HIV infectivity. These mutations map to the CA CTD and to spacer peptide 1 (SP1), the portion of the precursor Gag protein immediately C terminal to the CTD. The compensatory mutations do not locate to the molecularly modeled intermolecular NTD-CTD interface. Rather, the compensatory mutations appear to act indirectly, possibly by realignment of the C-terminal helix of the CA CTD, which participates in the NTD-CTD interface and has been shown to serve an important role in the assembly of infectious virus.