Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7).

BACKGROUND: Recently, we reported a novel oligoguanidine transporter system, polyarginine (R(7)), which, when conjugated to spectroscopic probes (e.g., fluorescein) and drugs (e.g., cyclosporin A), results in highly water-soluble conjugates that rapidly enter cells and tissues. We report herein the preparation of the first R(7) peptide conjugates and a study of their cellular and organ uptake and functional activity. The octapeptide (psi)(epsilon)RACK was selected for this study as it is known to exhibit selective epsilon protein kinase C isozyme agonist activity and to reduce ischemia-induced damage in cardiomyocytes. However, (psi)(epsilon)RACK is not cell-permeable. RESULTS: Here we show that an R(7)-(psi)(epsilon)RACK conjugate readily enters cardiomyocytes, significantly outperforming (psi)(epsilon)RACK conjugates of the transporters derived from HIV Tat and from Antennapedia. Moreover, R(7)-(psi)(epsilon)RACK conjugate reduced ischemic damage when delivered into intact hearts either prior to or after the ischemic insult. CONCLUSIONS: Our data suggest that R(7) converts a peptide lead into a potential therapeutic agent for the ischemic heart.

A new monofluorinated phosphatidylcholine forms interdigitated bilayers.

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.

Stress and nursing in the pig: role of HPA axis and endogenous opioid peptides.

To determine the effect of stress on nursing, and the roles of HPA activity and opioid peptides, nine sows had their piglets removed for 2 h and were treated as follows: (a) control; (b) nose-snare restraint for 20 min; (c) naloxone injections (i.v. 2 mg/kg); and (d) snare + naloxone. After the treatment, the piglets were returned, milk ejections were timed, and the sows' blood sampled every 10 min for cortisol, growth hormone (GH), and prolactin assays. Piglet removal increased cortisol and decreased prolactin and GH. This was reversed when the piglets were returned. Restraint increased cortisol and decreased GH, but did not affect prolactin. Naloxone alone increased cortisol and decreased GH but did not increase the effect of restraint. The rise in GH following the piglets' return was abolished by the combination of restraint and naloxone. Neither restraint nor naloxone delayed the latency to first milk ejection or reduced the frequency. No unsuccessful nursings were observed. First milk ejections occurred when cortisol levels were elevated. Stress-induced activity of the hypothalamo-pituitary-adrenocortical (HPA) axis does not inhibit milk ejection in the pig, but this is not due to a protective opioid action. Endogenous opioids protect lactogenic hormones against inhibition by stress.

Effect of miconazole and clotrimazole on K+ release and inhibition of ergosterol biosynthesis in Trichophyton mentagrophytes and related ultrastructural observations.

Measurement of K+ ions in extracellular fluids was used to provide evidence of direct membrane damage induced by miconazole and clotrimazole in Trichophyton mentagrophytes mycelium. K+ release from mycelium treated with high concentrations of drug was extensive, although leakage occurred more rapidly and to a greater extent with miconazole than with clotrimazole. Cells treated with fungicidal concentrations of drug were completely necrotic. The effect of miconazole was concentration dependent. A reduction in ergosterol levels was evident after 5 h treatment with miconazole. Further reduction in ergosterol concentration was demonstrated after 24 h with fungistatic concentrations of drug and was a concentration dependent effect. Fungistatic concentrations of miconazole also induced ultrastructural changes in mycelium, which were apparent within 5 h of treatment and became more pronounced after 24 h. Clotrimazole also reduced ergosterol levels and affected ultrastructure of mycelium. The plasma membrane in particular was affected by both these drugs.

Ultrastructure of protoplasts from mycelium and microconidia of Trichophyton mentagrophytes.

Protoplast formation from mycelium and microconidia of Trichophyton mentagrophytes was achieved with Novozym 234. Pretreatment procedures with dithiothreitol or urea mercaptoethanol sodium lauryl sulphate before digestion with Novozym 234 greatly reduced protoplast yield from mycelium. Snail gut enzyme did not protoplasts in good yield. Scanning electron microscopy of mycelium protoplasts showed the acquired spherical shape. The plasma membrane appeared finely granular although remnants of cell wall could sometimes be observed. Transmission electron microscopy showed the cell interior of these protoplasts was plasmolysed. Microconidia treated with Novozym 234 displayed a range of cell wall digestion, with intact protoplasts showing distinct cytoplasmic organelles.

The effect of imidazoles on germination of arthrospores and microconidia of Trichophyton mentagrophytes.

The effects of imidazole antifungal drugs on germination of Trichophyton mentagrophytes microconidia and arthrospores were investigated. A Coulter Counter was used to estimate spore swelling and microscopy to assess germ-tube formation. Both spore forms germinated in SLM, pH 6, at 32 degrees C and these conditions were used as a control. The inhibition of spore germination by imidazoles was concentration dependent and required higher drug levels than MICs. There was good correlation between reduction in swelling and inhibition of germ-tube formation. In order to produce greater than 90% reduction of germ-tube formation higher concentrations of imidazole were necessary for arthrospores than for microconidia. There was no clear difference in potency of imidazoles except for ketoconazole which was markedly less effective. No increase in sensitivity of microconidia to miconazole was observed with time of addition during the initial 3 h of germination. Although the inhibitory effect of miconazole on swelling of micronidia was partially reduced in the presence of Mg++, germ-tube formation was not altered.

Spore differentiation in a clinical strain of Trichophyton mentagrophytes.

Spore differentiation and, in particular, arthrosporogenesis in a clinical strain of T. mentagrophytes was investigated using a variety of methods and by altering environmental conditions. Results are discussed with reference to the in vivo situation. Arthrospores were obtained in the presence of increased CO2 tension but not increased N2 tension. High humidity was necessary for arthrospore formation but maturity (i.e. crops of single spores) was associated with conditions of reduced humidity. Desiccation reduced arthrospore viability. Glucose and peptone based media were suitable for arthrospore formation. Arthrospores were produced at 30 degrees C and 37 degrees C, but 30 degrees C is preferred since chlamydospores were prevalent at 37 degrees C. Conditions for production of arthrospore, microconidial and mycelial suspensions are presented.

The sensitivity of mycelium, arthrospores and microconidia of Trichophyton mentagrophytes to imidazoles determined by in-vitro tests.

With mycelium, arthrospores and microconidia of trichophyton mentagrophytes as inocula, a variety of in-vitro tests were used to assess the antifungal activity of the imidazoles; miconazole, clotrimazole, ketoconazole, econazole and tioconazole. For mycelial sensitivity, an agar plus method, a microtitre method using fragmented mycelial suspension and a turbidometric method were employed to determine fungistatic effects while a mycelial plug method indicated fungicidal activity. Spore susceptibility was determined by broth dilution and agar dilution methods for fungistatic action, while fungicidal activity was determined by measurement of rate of kill. The results obtained were affected to varying degrees by the test procedure, temperature and time of incubation, medium, pH and solvent. The spore forms were not more resistant than mycelium to the fungistatic effects of the imidazoles. There was little to choose between the various imidazoles in respect to their performance in these tests, with the exception of ketoconazole, which consistently gave higher MICs.

Energetics and possible mechanisms of ion-beam protein tritiation based on AB initio potential surfaces.

Ab initio self-consistent field potential energy surfaces for the approach of T, T2, T+, T3+ and HeT+ to glycine in the gas phase have been determined and this data used to obtain insight into mechanisms of experimental ion-beam protein tritiation processes. Results of these calculations show that the ionic species T+, T3+ and HeT+ can form stable adducts with glycine (Gly) and that each functions as a tritiation agent forming the complex GlyT+. Neutral T and T2 experience a purely repulsive interaction with Gly and do not form an intermediate complex. These neutral species are expected to be less effective tritiation agents than the respective ions, in agreement with experimental observations. The fate of the stable GlyT+ complex is discussed and it is proposed that this species is neutralized by electron capture to give GlyT which spontaneously dissociates to either Gly+T or tritiated glycine (Gly*)+H, with the latter reaction product channel favored statistically. The most likely site of exchange is predicted to be at the amine nitrogen although significance exchange is expected to occur at the alpha-carbon site by a somewhat more complex reaction mechanism.

The extraction of lymphoid cells from the human nasopharynx.

No criteria exist at present for judging the quality, extent, specificity, immunological memory or degree of functional autonomy of the local mucosal immune response in humans owing to difficulty in obtaining suitable specimens for assay. This paper offers a simple, practical, atraumatic technique for the extraction of viable lymphoid cells from the human nasopharyngeal mucosa together with some preliminary figures on the numbers of cells obtained and lymphocyte responses to mitogenic stimuli. The technique is based on the collection of nasal washings with chilled saline and sufficient lymphoid cells have been collected by this method to make multiple in vitro cultures. Cultures have been maintained for over 5 days and significant blastogenic responses have been recorded suggesting that the luminal cells retain some degree of functional competence.