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Peptides are emerging as a new tool in drug and gene delivery. Peptide-drug conjugates and peptide-modified drug delivery systems provide new opportunities to avoid macrophage recognition and subsequent phagocytosis, cross endothelial and epithelial barriers, and enter the cytoplasm of target cells. Peptides are relatively small, low-cost, and are stable in a wide range of biological conditions. In this review, we summarize recent work in designing peptides to enhance penetration of biological barriers, increase cell uptake, and avoid the immune system. We highlight recent successes and contradictory results, and outline common emerging concepts and design rules. The development of sequence-structure-function relationships and standard protocols for benchmarking will be a key to progress in the field.

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New apatite (AP)/nanodiamond (ND) coating has been developed to improve physical and biological properties of stainless steel (SS) versus single AP coating. Homogeneously electrodeposited AP-ND layer demonstrates increased mechanical strength, interlayer cohesion and ductility. In the absence of serum, osteoblast-like MG63 cells attach well but poorly spread on both AP and AP-ND substrata. Pre-adsorption with serum or fibronectin (FN) improves the cellular interaction-an effect that is better pronounced on the AP-ND coating. In single protein adsorption study fluorescein isothiocyanate-labeled FN (FITC-FN) shows enhanced deposition on the AP-ND layer consistent with the significantly improved cell adhesion, spreading and focal adhesions formation (in comparison to SS and AP), particularly at low FN adsorption concentrations (1 µg/ml). Higher FN concentrations (20 µg/ml) abolish this difference suggesting that the promoted cellular interaction of serum (where FN is low) is caused by the greater affinity for FN. Moreover, it is found that MG63 cells tend to rearrange both adsorbed and secreted FN on the AP-ND layer suggesting facilitated FN matrix formation.

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This 81 years old man with chronic obstructive pulmonary disease (COPD), chronic sepsis, chronic renal failure dependent on mechanical ventilation presented in the course of treatment with massive lower gastrointestinal bleeding (LGIB). Selective angiography of inferior mesenteric artery was performed 18 hours after first bleeding and localized source of bleeding at the distal colon as a contrast in the lumen of the gut. Direct intraarterial injection of 3.4 micrograms Vasopressin was carried out in inferior mesentery artery for preparation of surgery. During surgery the colonoscopy was done and despite of the high operative risk total colectomy with ileostomy was performed. This case confirms that there are not alternatives of colectomy in continuing LGIB from colonic diverticula even in the high risk patients.

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AIMS: To evaluate factors potentially contributing to the long-term persistence of Salmonella enterica serovar Enteritidis phage type (PT) 30 in an almond orchard. METHODS AND RESULTS: Surface and subsurface soil temperatures, and air temperatures in a radiation shelter, were recorded during a 12-month period, and were used to identify relevant storage temperatures (20 or 35 degrees C) for microcosms of two different soil types (clay and sandy loams) with moisture levels near saturation or near field capacity. Salmonella Enteritidis PT 30 was inoculated into the microcosms at 6 log CFU g(-1) dry weight. Between 14 and 180 days of incubation, counts of S. Enteritidis PT 30 decreased rapidly at 35 degrees C and were significantly different (P < 0.05) from counts at 20 degrees C, regardless of the soil type or moisture level. Salmonella was detected by enrichment of 10-g samples from all microcosms after 180 days of incubation at 20 degrees C, but from none of the microcosms held at 35 degrees C. To measure the potential for the growth of S. Enteritidis PT 30 in clay loam soil, an aqueous extract of almond hulls (containing 1.6% mono and disaccharides) or equivalent volume of water was added 7 days after inoculation. Significant (P < 0.05) growth of S. Enteritidis PT 30 was observed within 8 or 24 h of adding hull extract, but not water, to soil. CONCLUSIONS: Opportunities may exist for S. Enteritidis PT 30 to survive for an extended time in almond orchard soils and to grow in these soils where hull nutrients are released. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature has a significant impact on the long-term survival of S. Enteritidis PT 30 in soil, and nutrients leached from almond hulls may result in Salmonella growth. These factors should be considered in the design of Good Agricultural Practices for almonds.

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Arbuscular mycorrhizal fungi (AMF) are potentially important in nutrient cycling in agricultural soils and particularly in soils managed for organic production; little is known, however, about the interrelationships between AMF and other members of soil microbial communities. Ammonia oxidizing bacteria (AOB) are a trophic group of bacteria having an enormous impact on nitrogen availability in soils and are expected to be influenced by the presence of AMF. In a field study, we utilized a unique genetic system comprised of a mycorrhiza defective tomato mutant (named rmc) and its mycorrhiza wild-type progenitor (named 76RMYC+). We examined the effect of AMF by comparing AOB community composition and populations in soil containing roots of the two tomato genotypes in an organically managed soil. Responses of AOB to soil N and P amendments were also studied in the same experiment. Phylogenetic analysis of cloned AOB sequences, derived from excised denaturing gradient gel electrophoresis (DGGE) bands, revealed that the organic farm soil supported a diverse yet stable AOB community, which was neither influenced by mycorrhizal colonization of roots nor by N and P addition to the soil. Real-time TaqMan polymerase chain reaction (PCR) was used to quantify AOB population sizes and showed no difference between any of the treatments. An alternative real-time PCR protocol for quantification of AOB utilizing SYBR green yielded similar results as the TaqMan real-time PCR method, although with slightly lower resolution. This alternative method is advantageous in not requiring the detailed background information about AOB community composition required for adaptation of the TaqMan system for a new soil.

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Microbial communities in subsurface environments are poorly characterized and the impacts of anthropogenic contamination on their structure and function have not been adequately addressed. The release of contaminant(s) to a previously unexposed environment is often hypothesized to decrease the diversity of the affected community. We characterized the structure of microbial communities along a gradient of benzene, toluene, ethylbenzene, and xylene (BTEX) and methyl-tert-butyl-ether (MTBE) contamination, resulting from a petroleum spill, within a shallow sandy aquifer at Vandenberg Air Force Base (VAFB) in Lompoc, CA. Differences in microbial community composition along the contaminant plume were assessed via a combinatorial approach utilizing denaturing gradient gel electrophoresis (DGGE), cloning and sequencing, intergenic transcribed spacer analysis (ITS), and comparative phylogenetic analysis of partial 16S rDNA sequences. Substantial bacterial sequence diversity, similar levels of species richness, and similar phylo-groups (including the Cytophaga-Flavobacterium-Bacteroidetes group and numerous members of the alpha-, beta-, gamma-, delta-, and epsilon-groups of the proteobacteria) were observed in both uncontaminated and contaminated regions of the aquifer. High-resolution measures (ITS fingerprinting and phylogenetic inference) readily separated communities impacted by the original petroleum spill (in source zone) from those in other parts of the aquifer and indicated that communities exposed to MTBE only were similar to communities in uncontaminated regions. Collectively, these data suggest that petroleum contamination alters microbial community structure at the species and subspecies level. Further study is required to determine whether these changes have an impact on the functioning of this subsurface ecosystem.

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We show that the peptide backbone of an alpha-helix places a severe thermodynamic constraint on transmembrane (TM) stability. Neglect of this constraint by commonly used hydrophobicity scales underlies the notorious uncertainty of TM helix prediction by sliding-window hydropathy plots of membrane protein (MP) amino acid sequences. We find that an experiment-based whole-residue hydropathy scale (WW scale), which includes the backbone constraint, identifies TM helices of membrane proteins with an accuracy greater than 99 %. Furthermore, it correctly predicts the minimum hydrophobicity required for stable single-helix TM insertion observed in Escherichia coli. In order to improve membrane protein topology prediction further, we introduce the augmented WW (aWW) scale, which accounts for the energetics of salt-bridge formation. An important issue for genomic analysis is the ability of the hydropathy plot method to distinguish membrane from soluble proteins. We find that the method falsely predicts 17 to 43 % of a set of soluble proteins to be MPs, depending upon the hydropathy scale used.

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The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C(T)) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.

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The reliability of the transmembrane (TM) sequence assignments for membrane proteins (MPs) in standard sequence databases is uncertain because the vast majority are based on hydropathy plots. A database of MPs with dependable assignments is necessary for developing new computational tools for the prediction of MP structure. We have therefore created MPtopo, a database of MPs whose topologies have been verified experimentally by means of crystallography, gene fusion, and other methods. Tests using MPtopo strongly validated four existing MP topology-prediction algorithms. MPtopo is freely available over the internet and can be queried by means of an SQL-based search engine.

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Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin's exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

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Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5'-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.

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Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in soil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.

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The amphipathic alpha-helix is a recurrent feature of membrane-active proteins, peptides, and toxins. Despite extensive biophysical studies, the structural details of its affinity for membrane interfaces remain rather vague. We report here the first results of an effort to obtain detailed structural information about alpha-helices in membranes by means of a novel X-ray diffraction method. Specifically, we determined the transbilayer position and orientation of an archetypal class A amphipathic helical peptide in oriented fluid-state dioleoylphosphatidylcholine (DOPC) bilayers. The peptide, Ac-18A-NH2(Ac-DWLKAFYDKVAEKLKEAF-NH2), is a model for class A amphipathic helices of apolipoprotein A-I and other exchangeable lipoproteins. The diffraction method relies upon experimental determinations of absolute scattering-length density profiles along the bilayer normal and the transbilayer distribution of the DOPC double bonds by means of specific bromination, and molecular modeling of the perturbed lipid bilayer (derived using the transbilayer distribution of the double bonds) and the peptide. The diffraction results showed that Ac-18A-NH2was located in the bilayer interface and that its transbilayer distribution could be described by a Gaussian function with a 1/e-halfwidth of 4.5(+/-0.3) A located 17.1(+/-0.3) A from the bilayer center, close to the glycerol moiety. Molecular modeling suggested that Ac-18A-NH2is helical and oriented generally parallel with the bilayer plane. The helicity and orientation were confirmed by oriented circular dichroism measurements. The width of the Gaussian distribution, a measure of the diameter of the helix, indicated that the Ac-18A-NH2helix penetrated the hydrocarbon core to about the level of the DOPC double bonds. Bilayer perturbations caused by Ac-18A-NH2were surprisingly modest, consisting of a slight decrease in bilayer thickness with a concomitant shift of the double-bond distribution toward the bilayer center, as expected from a small increase in lipid-specific area caused by the peptide.

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Changes in the structure of the hydrocarbon core (HC) of fluid lipid bilayers can reveal how bilayers respond to the partitioning of peptides and other solutes (Jacobs, R. E., and S. H. White. 1989. Biochemistry. 28:3421-3437). The structure of the HC of dioleoylphosphocholine (DOPC) bilayers can be determined from the transbilayer distribution of the double-bonds (Wiener, M. C., and S. H. White. 1992. Biophys. J. 61:434-447). This distribution, representing the time-averaged projection of the double-bond positions onto the bilayer normal (z), can be obtained by means of neutron diffraction and double-bond specific deuteration (Wiener, M. C., G. I. King, and S. H. White. 1991. Biophys. J. 60:568-576). For fully resolved bilayer profiles, a close approximation of the distribution could be obtained by x-ray diffraction and isomorphous bromine labeling at the double-bonds of the DOPC sn-2 acyl chain (Wiener, M. C., and S. H. White. 1991. Biochemistry. 30:6997-7008). We have modified the bromine-labeling approach in a manner that permits determination of the distribution in under-resolved bilayer profiles observed at high water contents. We used this new method to determine the transbilayer distribution of the double-bond bromine labels of DOPC over a hydration range of 5.4 to 16 waters per lipid, which reveals how the HC structure changes with hydration. We found that the transbilayer distributions of the bromines can be described by a pair of Gaussians of 1/e half-width A(Br) located at z = +Z(Br) relative to the bilayer center. For hydrations from 5.4 waters up to 9.4 waters per lipid, Z(Br) decreases from 7.97 +/- 0.27 A to 6.59 +/- 0.15 A, while A(Br) increased from 4.62 +/- 0.62 A to 5.92 +/- 0.37 A, consistent with the expected hydration-induced decrease in HC thickness and increase in area per lipid. After the phosphocholine hydration shell was filled at approximately 12 waters per lipid, we observed a shift in Z(Br) to approximately 7.3 A, indicative of a distinct structural change upon completion of the hydration shell. For hydrations of 12-16 waters per lipid, the bromine distribution remains constant at Z(Br) = 7.33 +/- 0.25 A and A(Br) = 5.35 +/- 0.5 A. The absolute-scale structure factors obtained in the experiments provided an opportunity to test the so-called fluid-minus method of structure-factor scaling. We found that the method is quite satisfactory for determining the phases of structure factors, but not their absolute values.

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Beta-sheets, in the form of the beta-barrel folding motif, are found in several constitutive membrane proteins (porins) and in several microbial toxins that assemble on membranes to form oligomeric transmembrane channels. We report here a first step towards understanding the principles of beta-sheet formation in membranes. In particular, we describe the properties of a simple hydrophobic hexapeptide, acetyl-Trp-Leu5 (AcWL5), that assembles cooperatively into beta-sheet aggregates upon partitioning into lipid bilayer membranes from the aqueous phase where the peptide is strictly monomeric and random coil. The aggregates, containing 10 to 20 monomers, undergo a relatively sharp and reversible thermal unfolding at approximately 60 degreesC. No pores are formed by the aggregates, but they do induce graded leakage of vesicle contents at very high peptide to lipid ratios. Because beta-sheet structure is not observed when the peptide is dissolved in n-octanol, trifluoroethanol or sodium dodecyl sulfate micelles, aggregation into beta-sheets appears to be an exclusive property of the peptide in the bilayer membrane interface. This is an expected consequence of the hypothesis that a reduction in the free energy of partitioning of peptide bonds caused by hydrogen bonding drives secondary structure formation in membrane interfaces. But, other features of interfacial partitioning, such as side-chain interactions and reduction of dimensionality, must also contribute. We estimate from our partitioning data that the free energy reduction per residue for aggregation is about 0.5 kcal mol-1. Although modest, its aggregate effect on the free energy of assembling beta-sheet proteins can be huge. This surprising finding, that a simple hydrophobic hexapeptide readily assembles into oligomeric beta-sheets in membranes, reveals the potent ability of membranes to promote secondary structure in peptides, and shows that the formation of beta-sheets in membranes is more facile than expected. Furthermore, it provides a basis for understanding the observation that membranes promote self-association of beta-amyloid peptides. AcWL5 and related peptides thus provide a good starting point for designing peptide models for exploring the principles of beta-sheet formation in membranes.

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We have examined the interactions of the six known rabbit neutrophil defensin antimicrobial peptides with large unilamellar vesicles (LUV) made from various lipid mixtures based on the lipid composition of Escherichia coli membranes. We find that the permeabilization of LUV made from E. coli whole lipid extracts differs dramatically from that of single-component LUV made from palmitoyl-oleoyl-phosphatidylglycerol (POPG). Specifically, defensins NP-1, NP-2, NP-3A, NP-3B, and a natural mixture of the six defensins cause fast nonpreferential leakage of high molecular weight dextrans as well as the low molecular weight fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) from E. coli whole lipid LUV through large, transient membrane lesions. In contrast, release of ANTS/DPX from POPG LUV induced by the defensins is slow and graded with preference for DPX (Hristova, K., Selsted, M. E., and White, S. H. (1996) Biochemistry 35, 11888-11894). Interestingly, defensins NP-4 and NP-5 alone do not induce leakage from E. coli whole lipid LUV, whereas only NP-4 is ineffective with POPG LUV. Examination of the sequences of the six defensins suggests that the inactivity of NP-4 and NP-5 may be due to their lower net positive charge and/or the substitution of a Thr for the Arg or Lys that follows the fourth Cys residue. We found the presence of three major lipid components of E. coli whole lipid to be essential for creation of the large lesions observed in LUV: phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Cardiolipin appears to play a key role because no leakage can be induced when only phosphatidylglycerol and phosphatidylethanolamine are present. These results indicate the importance of membrane lipid composition in the permeabilization of cell membranes by rabbit defensins.

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Human antimicrobial neutrophil defensin HNP-2 has been shown to form large multimeric pores in pure 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) bilayers that lead to all-or-none release of vesicle contents [Wimley et al. (1994) Protein Sci.3, 1362-1373]. Because human neutrophil defensins form natural dimers in solution, the question arises as to the role of dimerization in pore formation. However, the dimers are so stable that this question is not easily answered directly. Rabbit neutrophil defensins, whose three-dimensional structures are very similar to those of human defensins, are monomeric in aqueous solution and thus provide an opportunity to test the hypothesis that dimerization may play a role in multimeric pore formation. We therefore examined the interactions of the six known rabbit neutrophil defensins with large unilamellar vesicles (LUV) under the conditions known to lead to stable pore formation by HNP-2. We find that the rabbit defensins bind strongly to LUVs formed from pure POPG or mixtures of POPG with neutral (zwitterionic) phospholipid but induce leakage of vesicle contents only from pure POPG vesicles. Rabbit defensin NP-4 does not cause leakage under any conditions examined. The remaining defensins, NP-1, NP-2, NP-3A, NP-3B, and NP-5, cause graded release of the contents of pure POPG vesicles as does a mixture of the six defensins. The graded release indicates that the rabbit defensins do not form stable pores in the membrane. This result thus suggests that the structural features of human defensins that permit dimer formation in aqueous solution are likely to be important in the formation of multimeric pores.

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