We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Three analogues of LH-RH in which Dextrarotatory amino acids were substituted for the Gly6, and two additional analogues in which the Leu7 residue was also modified, were subjected to enzymic preparations derived from rat hypothalamus or anterior pituitary. These enzymes, known to cleave LH-RH, preferentially at the Gly6-Leu7 position, proved less effective in degrading all the analogues tested. Among the Gly6 substituted analogues, [ D-Trp6] LH-RH, having the highest LH-releasing activity, was most resistant to degradation. Additional modification, at position 7, although rendering the analogues immune to enzymic attack, did not further enhance their biological potency. These data suggest that degradation of LH-RH is a physiological determinant of its biological activity and has therefore to be considered with on designing new, potent analogues of the hormone.

The in vitro antiviral activity of two amphiphilic synthetic peptides, modelin‐1 (mod‐1) and modelin‐5 (mod‐5), and of the natural antibacterial peptide magainin‐2 (mag‐2) against herpes simplex viruses type 1 (HSV‐1) and 2 (HSV‐2) were evaluated. The peptides were incubated with the virus, i.e. direct inactivation, and their effects examined by means of plaque reduction assay and/or reduction in virus yield. Only mod‐ displayed a strong antiviral effect against HSV‐1 and HSV‐2, with 50% effective dose (ED50) values of 4.6 and 4.1 μg/mL, respectively. Mag‐2, mod‐5 and a mixture of both had no significant inhibitory effect. Addition of mod‐1 up to a concentration of 100μg/mL to the culture medium had no significant cytotoxic effect on host vero cells, as measured by the trypan blue‐exclusion method. It showed, however, considerable hemolytic activity against human red blood cells. Experiments including acyclovir (ACV) as a reference viral inhibitor indicated that the mode of action of mod‐1 is different from that of ACV. In contrast to ACV, the peptide inactivates the virus following a very short incubation before vero cell infection, suggesting some kind of direct interaction of the peptide with the viral envelope, rather than inhibition of viral DNA replication or gene expression. Our results suggest that mod‐1 may be an effective topical antiviral agent against herpes viruses.

The hydrolysis of p-nitrophenylacetate has been studied between pH 6 and 9 in 3.3O/, (v/v) dioxane-0.1 M phosphate or Tris buffer. The results indicate that the hydrolysis is acid-catalyzed, base-catalyzed and has a pH-independent rate component. The observed rate constants for the hydrolysis were determined spectrophotometrically from the initial rate of appearance of p-nitrophenol (at 320 nm), p-nitrophenoxide (at 400 nm) and the initial rate of disappearance of p-nitrophenylacetate (at 270 nm). An equation has been derived permitting the calculation of first and second-order rate constants from the initial slopes of product appearance or reactant disappearance. The semilog method of first-order kinetic analyses could not be used since the hydrolysis of p-nitrophenylacetate did not go to completion.The apparent incomplete reaction is attributed to the establishment of an equilibrium between p-nitrophenol and p-nitrophenyl esters. The latter probably includes p-nitrophenyl phosphate. The rate constant for the synthesis of p-nitrophenyl esters has been determined and it also has acid, base and pH-independent catalytic components.The hydrolysis of an ester may occur through base-catalyzed, pH-independent and acid-catalyzed reaction pathways [l]. Hine [2] has demonstrated that these reaction pathways are independent of each other. Thus, the observed pseudo-first-order rate constant for the hydrolysis of the ester is made up of the sum of products of the catalyst concentration and a second-order rate constant. The latter rate constant represents the inherent ability of the catalyst to catalyze the reaction. In distilled water, the observed rate constant (krter) may be expressed as observed pseudo-first-order rate constant may be described as [4-71: (2)Yates and McClelland [8] have shown that in 1501, aqueous sulfuric acid, the mechanism of p-nitrophenylacetate hydrolysis is second order, acid-catalyzed with acyl-cleavage [9]. This may be expressed as:We present evidence here that acid-catalyzed pnitrophenylacetate hydrolysis may also occur a t neutral pH.The hydrolysis of p-nitrophenylacetate is generally followed spectrophotometrically a t 270 nm [8] for p-nitrophenylacetate loss or 400 nm for p-nitrophenoxide ion appearance or 300nm for p-nitrophenol appearance [lo]. The spectroscopic data may yield rate constants [10,11]. To obtain kTter, the procedure has been to determine the hydrolytic rate constants, k, in the presence of varying concentrations of catalyst and then to extrapolate the k krter = kHeO + OH- [OH-]. ver = k H~O + k~+ [H30+].Eur. J. Biochem. 41 (1974)

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.