[Specifity of optic disc evaluation in healthy subjects with large optic discs and physiologic cupping using confocal scanning laser ophthalmoscopy]. / Spezifität der konfokalen Scanning Laser Ophthalmoskopie bei großen Sehnerven mit physiologischer Exkavation.

PURPOSE: Imaging methods of the optic nerve head appear to have an increasing impact in glaucoma diagnosis. The aim of this study is to evaluate the specifity of the Heidelberg Retina Tomograph (software version 1.7 and 3.0) in subjects with physiological cupping and large optic discs. PATIENTS AND METHODS: 27 eyes of 27 subjects (mean age 41.3 ± 15.8 years) with bilateral physiological cupping and large optic discs (vertical cup-to-disc-ratio > 0.3, optic disc area 2.48 ± 0.45 mm2, max. 3.54 mm2) were included in a clinical study. All subjects had an intraocular pressure < 22 mmHg, physiological cupping by funduscopy and no nerve fibre layer defects (Scanning Laser Ophthalmoscope, Rodenstock, Germany). Standard achromatic perimetry (Humphrey Field Analyzer, Humphrey-Zeiss, 24/2 SITA or full threshold), short-wavelength automated perimetry (Humphrey Field Analyzer, Humphrey-Zeiss), and frequency doubling technology (FDT, Humphrey-Zeiss) revealed no visual field defects. Optic disc imaging was performed in all subjects using the Heidelberg Retina Tomograph II (HRT). Optic disc images were transferred to the software-update of the HRT 3 (Version 3.0, Heidelberg Engineering). Specifity was calculated using the Moorfields regression analysis (MRA, software version 1.7 and 3.0) and the glaucoma probability score (GPS analysis) using all disc sectors and omitting the nasal and 3 nasal sectors. RESULTS: Specifity of the MRA (software version 1.7) was 66.6 % (most specific criteria), and 22.2 % (least specific criteria). Specifity of the MRA (software version 3.0) was 33.3 % (most specific criteria), and 14.8 % (least specific criteria), whereas specifity of the GPS analysis was 37.0 % (most specific criteria), and 11.1 % (least specific criteria). When the nasal sectors were omitted for analysis, specifity increased for the MRA analysis, but not for the GPS analysis. CONCLUSIONS: Specifity of the MRA was unsatisfactory using the software version 1.7 and 3.0 in subjects with large optic discs and physiological cupping when the nasal sectors were included in the analysis. The observer-independent GPS analysis did not improve the results in these subjects.

Aproximación teórica del profesional enfermero a los dispositivos legales para afrontar la violencia de género / Theoretical professional nurssing approach to the legal devices state gives to face gender violence

La violencia contra la mujer ha constituido un fenómeno invisible durante décadas. Incluso hoy continúa siendo difícil de identificar. Así, la II Conferencia Mundial sobre la Condición Jurídica y Social de la Mujer celebrada en 1980 en Copenhague ya se refería a este tipo de maltrato como "el crimen más silenciado del mundo". Nosotros, como enfermeros guiados por conocimientos legales, éticos y deontológicos, y junto a otros profesionales, nos percatamos de la inmensa importancia que tiene la defensa de los derechos humanos y por ello creemos oportuna la realización de este estudio en el que intentaremos resumir los distintos sistemas de protección estatales para satisfacer nuestro objetivo último: acercar al profesional enfermero que lea estas líneas, al conocimiento de dichos sistemas y al contexto de la violencia de género. La técnica empleada en esta revisión bibliográfica es la de análisis de contenido, en la modalidad temática, que se basa en la lectura como instrumento de recogida de datos; lectura que debe realizarse siguiendo el método científico, es decir, debe ser sistemática, objetiva, replicable y válida. Todos estos dispositivos de protección estatales son relevantes pero no debemos de olvidar uno en absoluto desdeñable: el equipo interdisciplinar sanitario entre el que se incluye el conjunto de Enfermería que, al conocer dichos dispositivos, puede convertirse en un aliado destacado a la hora de garantizar la seguridad e integridad de la mujer (AU)

Violence against women has been an unseen phenomenon for decades. Even today it remains being difficult to identify. Thus, In the Second World Conference about the Status of women celebrated in Copenhagen in 1980 referred this type of abuse as "the most silenced crime in the world". We, as nurses guided by legal, ethical and deontological knowledge, and with other professionals, notice the immense importance of defending the Human Rights and because of that, this issue is the reason for this study in which we try to summarize the various State Protection Systems in order to get our ultimate goal: to approach the professional nurse who reads these lines to the knowledge of these systems and the knowledge of the context of gender violence. The technique used in this review is a content analysis procedure, within its thematic category which is based on reading as a tool of data collection; reading that has to be done following the scientific method, i.e. it must be systematic, objective and valid. All these State Protective devices are relevant, but we must remember one important aspect: the interdisciplinary health team which includes among the set of nursing that, realizing of this devices, can become in a prominent ally in women's ensuring security and integrity (AU)

[Color Doppler sonography of retrobulbar vessels and hypercapnia in normal tension glaucoma]. / Farbduplexsonographie der retrobulbären Gefäße und Hyperkapnie bei Normaldruckglaukom.

PURPOSE: The aim of the study was to investigate retrobulbar flow velocities during hypercapnia in patients with normal tension glaucoma (NTG) without systemic vasospasm and jn controls. METHODS: A total of 16 NTG patients (mean age 58 ± 14 years) and 16 control subjects (mean age 50 ± 13 years, p = 0.10) were enrolled in this study. Flow velocities, peak systolic velocity (PSV), end-diastolic velocity (EDV) and resistive indices (RI) of the ophthalmic (OA) and central retinal arteries (CRA) were assessed using color Doppler imaging. Blood flow velocities were measured under normocapnic and hypercapnic conditions (increasing the end-tidal pCO(2) by 15%). Blood pressure, ventilation rate and oxygen saturation were monitored simultaneously. RESULTS: During hypercapnia, velocity responses of the PSV (p = 0.044) and EDV (p = 0.037) of the CRA were significantly different in NTG patients and healthy controls, showing a greater increase of flow velocities in control subjects. Flow velocities of the OA increased significantly in both groups (PSV p = 0.039, EDV p = 0.003) during hypercapnia. Blood pressure, oxygen saturation and intraocular pressure changed similarly in both study groups with carbon dioxide provocation. CONCLUSIONS: Velocity response to hypercapnia was reduced in the CRA of NTG patients compared to controls. This may indicate a faulty vasodilatory response in NTG patients without vasospastic disease.

Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain.

BACKGROUND AND PURPOSE: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands. EXPERIMENTAL APPROACH: We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays. KEY RESULTS: Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did. CONCLUSIONS AND IMPLICATIONS: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

Opacity of electromagnetically induced transparency for quantum fluctuations.

We analyze the propagation of a pair of quantized fields inside a medium of three-level atoms in a Lambda configuration. We calculate the stationary quadrature noise spectrum of the field, in the case where the probe field is in a squeezed state and the atoms show electromagnetically induced transparency. We find an oscillatory transfer of the initial quantum properties between the probe and pump fields which is most strongly pronounced when both fields have comparable intensities. This implies that the quantum state measured after propagation can be completely different from the initial state, even though the mean values of the field are unaltered.

Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain.

BACKGROUND AND PURPOSE: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different. EXPERIMENTAL APPROACH: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay. KEY RESULTS: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. CONCLUSIONS AND IMPLICATIONS: The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).

Antimicrobial activity of arginine- and tryptophan-rich hexapeptides: the effects of aromatic clusters, D-amino acid substitution and cyclization.

Many antimicrobial peptides bear arginine (R)- and tryptophan (W)-rich sequence motifs. Based on the sequence Ac-RRWWRF-NH2, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, L-D-amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW-peptides displayed moderate activity towards Gram-positive Bacillus subtilis (15 < MIC < 31 microm) and were inactive against Gram-negative Escherichia coli at peptide concentrations < 100 microm. Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d-amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW-peptides at 100 microm concentration was low (< 7% lysis for linear R/W-rich peptides and < 28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high-performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.

Programa de formación en educación para la salud en el currículo enfermero / A health education program within the general degree in nursing curriculum

La educación para la salud en el marco de la promoción de la salud, la prevención y el tratamiento de los problemas de salud ha dejado de ser un aspecto deseable de la atención sanitaria, para convertirse en una actividad concreta, perfectamente explicitada y, a menudo fundamental, de los programas de salud. Es preciso, por tanto, que el currículo de diplomado en enfermería contemple un enfoque educativo en los cuidados enfermeros que se prestan a los individuos que padecen problemas de salud, y no se centren sólo en los aspectos clínicos individuales. Además de la incorporación de contenidos educativos de manera general en las materias del currículo base enfermero, es incuestionable considerar la aportación de una asignatura específica en materia de educación para la salud. Así, en este trabajo proponemos un Programa de Formación en Educación para la Salud como asignatura básica que ampliará el horizonte y las expectativas profesionales de los estudiantes de enfermería en lo que se refiere a la atención a las familias, a los grupos y a las comunidades, tanto fuera como dentro de los muros de las instituciones sanitarias, para acercarse hasta donde ellos vivan, se relacionen y trabajen (AU)

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

State reconstruction of the kicked rotor.

We propose two experimentally feasible methods based on atom interferometry to measure the quantum state of the kicked rotor.

Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation.

Human lumbar CSF patterns of Abeta peptides were analysed by urea-based beta-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Abeta-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Abeta1-37/38/39 was found in addition to Abeta1-40 and Abeta1-42. Remarkably, Abeta1-38 was present at a higher concentration than Abeta1-42, being the second prominent Abeta peptide species in CSF. Patients with Alzheimer's disease (AD, n = 12) and patients with chronic inflammatory CNS disease (CID, n = 10) were differentiated by unique CSF Abeta peptide patterns from patients with other neuropsychiatric diseases (OND, n = 37). This became evident only when we investigated the amount of Abeta peptides relative to their total Abeta peptide concentration (Abeta1-x%, fractional Abeta peptide pattern), which may reflect disease-specific gamma-secretase activities. Remarkably, patients with AD and CID shared elevated Abeta1-38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE epsilon4 alleles resulted in an overall reduction of CSF Abeta peptides, which was pronounced for Abeta1-42. The severity of dementia was significantly correlated to the fractional Abeta peptide pattern but not to the absolute Abeta peptide concentrations.

Phosphopeptide analysis by positive and negative ion matrix-assisted laser desorption/ionization mass spectrometry.

This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.

Optimization of the antimicrobial activity of magainin peptides by modification of charge.

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues.

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.

Role of helix formation for the retention of peptides in reversed-phase high-performance liquid chromatography.

In order to get insight into the role of helix formation for retention in reversed-phase HPLC, we have studied the isocratic retention behavior of amphipathic and non-amphipathic potentially helical model peptides. Plots of the logarithmic capacity factor in absence of organic solvent (ln k0) versus l/T were used to derive the enthalpy, deltaH0, the free energy, deltaG0, the entropy of interaction, deltaS0, and the heat capacity change, deltaCp. Retention of all peptides was accompanied by negative deltaCp revealing that hydrophobic interactions play a large role independent of peptide sequence and secondary structure. deltaH0 was negative for the amphipathic analogs and was attributed mainly to helix formation of these peptides upon interaction with the stationary phase. In contrast, deltaH0 was considerably less exothermic or even endothermic for the non-amphipathic analogs. The differences in helix formation between the individual analogs were quantified on the basis of thermodynamic data of helix formation previously derived for peptides in a hydrophobic environment. Correlation of the helicity with the free energy of stationary phase interaction revealed that helix formation accounts for approximately 40-70% of deltaG0, and is hence in addition to the hydrophobic effect a major driving force of retention.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin-1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein.

We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.

The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties.

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH(2) was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH(2) inhibited spontaneous locomotor activity in mice with ID(50) of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH(2) could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH(2), [Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-NH(2) ([F/G]NC(1-13)NH(2)) inhibited the noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH(2) in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH(2) and of [F/G]NC(1-13)NH(2), it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.

Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptides.

The cellular uptake of a peptide set derived from membrane-permeable alpha-helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity-dependent differences became apparent only after washing the peptide-loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 degrees C led to similar results, indicating the nonendocytic character of the uptake. With a view to practical applications, the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell-type specific vector peptides.

Comparison of the effects of 5- and 6-HOAt on model peptide coupling reactions relative to the cases for the 4- and 7-Isomers.

Synthesis of 5- and 6-HOAt has completed the full set of the four HOAt isomers derived from HOBt by insertion of a single nitrogen atom in the benzenoid nucleus. Comparison of the reactivity of all four isomers in model peptide coupling reactions has confirmed the unique character of the 7-isomer in promoting selectivity and maintaining configuration at the reactive carboxylic acid residue.