Reversible pegylation prolongs the hypotensive effect of atrial natriuretic peptide

Natriuretic peptides (NP), including atrial natriuretic peptide (ANP), induce potent natriuresis and vasodilation and thereby generate hypotension in vivo. Despite intensive efforts, clinical application of NP as an antihypertensive agent is limited because of their short biological half-life and poor bioavailability. Recently, we have developed a strategy that facilitates slow release of peptides from PEG-peptide inactive conjugates, based on reversible pegylation. Peptides prepared by this approach undergo slow, spontaneous chemical hydrolysis at physiological conditions, releasing the native active peptide/protein drug from the inactive conjugates over prolonged periods. A PEG chain of 30 kDa was linked covalently to the alpha-amino side chain of the hormone via a MAL-Fmoc-NHS spacer, yielding PEG 30-Fmoc-ANP, a prodrug that releases the native hormone upon incubation at physiological conditions. Bolus administration of native ANP to Wistar rats receiving adrenaline yields a short, transitory effect in lowering blood pressure (BP), reaching a maximum at 2 min, and then returning to control values after 12 to 25 min. In contrast, administration of PEG 30-Fmoc-ANP lowered BP following a lag period of 50 min, and maintained low BP for a period exceeding 60 min. Saline or PEG 30-Fmoc-Alanine were not effective in lowering BP in Wistar rats. These results show that the novel compound, PEG 30-Fmoc-ANP, is a reversible pegylated prodrug derivative that facilitates a prolonged BP lowering effect in rats and may be considered as a candidate for development into an antihypertensive drug.     

Altered GLUT4 translocation in skeletal muscle of 12/15-lipoxygenase knockout mice.

We have recently shown that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of actin microfilaments in rat cardiomyocytes, and that inhibition of 12-lipoxygenase abrogates insulin-stimulated GLUT4 translocation in these cells. In the present study, we used mice that were null for the leukocyte 12/15-lipoxygenase to explore the implications of this enzyme for insulin action under IN VIVO conditions. Insulin induced a profound reduction in blood glucose in both control and knockout mice. However, significantly higher serum insulin levels were observed in these animals. GLUT4 expression in heart and skeletal muscle was unaffected in KO mice. Insulin-regulated serine phosphorylation of Akt and GSK3alpha and GSK3beta was unaltered in heart and skeletal muscle of knockout mice, suggesting unaltered insulin signaling. Fractionation of hind limb muscles showed that insulin had induced a prominent translocation of GLUT4 to skeletal muscle plasma membranes in control mice. However, this response was largely reduced in knockout animals. Our data show that the lack of leukocyte 12/15-lipoxygenase does not lead to the development of an insulin-resistant phenotype. However, perturbation of GLUT4 translocation in skeletal muscle of knockout mice may indicate latent insulin resistance, and supports our hypothesis that eicosanoids are involved in insulin-mediated regulation of muscle glucose transport.     

Infection of CD127+ (interleukin-7 receptor+) CD4+ cells and overexpression of CTLA-4 are linked to loss of antigen-specific CD4 T cells during primary human immunodeficiency virus type 1 infection

We recently found that human immunodeficiency virus (HIV)-specific CD4+ T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+ T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+) T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+ lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+ cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+ T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+ T regulatory cells during PHI. However, 40% of CCR5+CD38+++ CD4+ T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+ T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+ T cells is associated with a combination of an infection of CCR5+ CD127+ memory CD4+ T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.     

Cell pairing and methylation in Tetrahymena thermophila are altered by exogenous homocysteine

Homocysteine is causally associated with birth defects such as spina bifida, and with premature vascular disease. We have investigated the effects of homocysteine on a cell-cell interaction in a fundamental eukaryotic system, the free-living ciliate Tetrahymena. Exogenously added homocysteine inhibits cell pairing in a dose-dependent manner. These effects are exacerbated by adenosine, which by itself has little demonstrable influence on pairing. S-adenosylhomocysteine (SAH) is a product of the reaction between adenosine and homocysteine, and is an inhibitor of methyl transferases. We therefore predicted that protein methylation would be significantly inhibited by homocysteine. A direct test of that hypothesis involved a demonstration that incorporation of an isotopically labeled methyl group from methionine into proteins was significantly reduced by homocysteine. The undermethylated proteins are of low molecular weight, and might correspond to known methylatable signaling proteins. We show that vanadate, an inhibitor of protein phosphatase, also inhibits cell pairing, and that the effects of vanadate and homocysteine are additive. This is the first demonstration that methylation and possibly phosphorylation play a regulatory role in cell-cell interactions in ciliates.     

Current status of molecular markers for early detection of sporadic pancreatic cancer

Pancreatic cancer (PC) is a highly lethal malignancy with near 100% mortality. This is in part due to the fact that most patients present with metastatic or locally advanced disease at the time of diagnosis. Significantly, in nearly 95% of PC patients there is neither an associated family history of PC nor of diseases known to be associated with an increased risk of PC. These groups of patients who comprise the bulk of PC cases are termed as "sporadic PC" in contrast to the familial PC cases that comprise only about 5% of all PCs. Given the insidious onset of the malignancy and its extreme resistance to chemo and radiotherapy, an abundance of research in recent years has focused on identifying biomarkers for the early detection of PC, specifically aiming at the sporadic PC cohort. However, while several studies have established that asymptomatic individuals with a positive family history of PC and those with certain heritable syndromes are candidates for PC screening, the role of screening in identifying sporadic PC is still an unsettled question. The present review attempts to assess this critical question by investigating the recent advances made in molecular markers with potential use in the early diagnosis of sporadic PC - the largest cohort of PC cases worldwide. It also outlines a novel yet simple risk factor based stratification system that could be potentially employed by clinicians to identify those individuals who are at an elevated risk for the development of sporadic PC and therefore candidates for screening.     

RGK family G-domain:GTP analog complex structures and nucleotide-binding properties

The RGK family of small G-proteins, including Rad, Gem, Rem1, and Rem2, is inducibly expressed in various mammalian tissues and interacts with voltage-dependent calcium channels and Rho kinase. Many questions remain regarding their physiological roles and molecular mechanism. Previous crystallographic studies reported RGK G-domain:guanosine di-phosphate structures. To test whether RGK proteins undergo a nucleotide-induced conformational change, we determined the crystallographic structures of Rad:GppNHp and Rem2:GppNHp to 1.7 and 1.8 Å resolutions, respectively. Also, we characterized the nucleotide-binding properties and conformations for Gem, Rad, and several structure-based mutants using fluorescence spectroscopy. The results suggest that RGK G-proteins may not behave as Ras-like canonical nucleotide-induced molecular switches. Further, the RGK proteins have differing structures and nucleotide-binding properties, which may have implications for their varied action on effectors.     

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.     

Nikkomycin production in pellets of Streptomyces tendae.

In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

Males that drop a sexually selected weapon grow larger testes

Costly sexually selected weapons are predicted to trade off with postcopulatory traits, such as testes. Although weapons can be important for achieving access to females, individuals of some species can permanently drop (i.e. autotomize) their weapons, without regeneration, to escape danger. We capitalized on this natural behavior to experimentally address whether the loss of a sexually selected weapon leads to increased testes investment in the leaf‐footed cactus bug, Narnia femorata Stål (Hemiptera: Coreidae). In a second experiment, we measured offspring production for males that lost a weapon during development. As predicted, males that dropped a hind limb during development grew significantly larger testes than the control treatments. Hind‐limb autotomy did not result in the enlargement of other nearby traits. Our results are the first to experimentally demonstrate that males compensate for natural weapon loss by investing more in testes. In a second experiment we found that females paired with males that lost a hind limb had 40% lower egg hatching success than females paired with intact males, perhaps because of lower mating receptivity to males with a lost limb. Importantly, in those cases where viable offspring were produced, males missing a hind limb produced 42% more offspring than males with intact limbs. These results suggest that the loss of a hind‐limb weapon can, in some cases, lead to greater fertilization success.     

Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.