Lipid selectivity in novel antimicrobial peptides: Implication on antimicrobial and hemolytic activity.

Antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. For an AMP to be considered as a therapeutic option, it must have not only potent antibacterial properties but also low hemolytic and cytotoxic activities [1]. Even though many studies have been conducted in order to correlate the antimicrobial activity with affinity toward model lipid membranes, the use of these membranes to explain cytotoxic effects (especially hemolysis) has been less explored. In this context, we studied lipid selectivity in two related novel AMPs, peptide 6 (P6) and peptide 6.2 (P6.2). Each peptide was designed from a previously reported AMP, and specific amino acid replacements were performed in an attempt to shift their hydrophobic moment or net charge. P6 showed no antimicrobial activity and high hemolytic activity, and P6.2 exhibited good antibacterial and low hemolytic activity. Using both peptides as a model we correlated the affinity toward membranes of different lipid composition and the antimicrobial and hemolytic activities. Our results from surface pressure and zeta potential assays showed that P6.2 exhibited a higher affinity and faster binding kinetic toward PG-containing membranes, while P6 showed this behavior for pure PC membranes. The final position and structure of P6.2 into the membrane showed an alpha-helix conversion, resulting in a parallel alignment with the Trps inserted into the membrane. On the other hand, the inability of P6 to adopt an amphipathic structure, plus its lower affinity toward PG-containing membranes seem to explain its poor antimicrobial activity. Regarding erythrocyte interactions, P6 showed the highest affinity toward erythrocyte membranes, resulting in an increased hemolytic activity. Overall, our data led us to conclude that affinity toward negatively charged lipids instead of zwitterionic ones seems to be a key factor that drives from hemolytic to antimicrobial activity.

Phenylalanine interaction with lipid monolayers at different pHs.

The influence of Phe on the surface pressure of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers at the air-water interface was studied at different initial surface pressures (26 and 40 mN/m) and two pHs (5.0 and 7.3) at constant temperature (20 °C). Changes produced by the aminoacid added to the subphase on the surface pressure and on the dipole potential of lipid monolayers were measured at a fixed area. Compressibility properties of the monolayers at different pHs were studied by (π-A) isotherms. The results suggest that Phe intercalates into a DPPC film at the air-water interface at pH 5 and forms a different arrangement at pH 7.3. The possible relevance of these results of the effect of Phe in physiological conditions is discussed.

Functional role of water in membranes updated: A tribute to Träuble.

The classical view of a cell membrane is as a hydrophobic slab in which only nonpolar solutes can dissolve and permeate. However, water-soluble non-electrolytes such as glycerol, erythritol, urea and others can permeate lipid membranes in the liquid crystalline state. Moreover, recently polar amino acid's penetration has been explained by means of molecular dynamics in which appearance of water pockets is postulated. According to Träuble (1971), water diffuses across the lipid membranes by occupying holes formed in the lipid matrix due to fluctuations of the acyl chain trans-gauche isomers. These holes, named "kinks" have the molecular dimension of CH2 vacancies. The condensation of kinks may form aqueous spaces into which molecular species of the size of low molecular weight can dissolve. This molecular view can explain permeability properties considering that water may be distributed along the hydrocarbon chains in the lipid matrix. The purpose of this review is to consolidate the mechanism anticipated by Träuble by discussing recent data in literature that directly correlates the molecular state of methylene groups of the lipids with the state of water in each of them. In addition, the structural properties of water near the lipid residues can be related with the water activity triggering kink formation by changes in the head group conformation that induces the propagation along the acyl chains and hence to the diffusion of water.

Study of surface damage on cell envelope assessed by AFM and flow cytometry of Lactobacillus plantarum exposed to ethanol and dehydration.

AIMS: In this work, we evaluated freeze-drying damage at the surface level of oenological strain Lactobacillus plantarum UNQLp155, as well as its ability to grow in a synthetic wine with and without pre-acclimation. METHODS AND RESULTS: Damage on cell surface was studied by flow cytometry, zeta potential and atomic force microscopy, and cell survival was analysed by plate count. Results showed that beside cells acclimated at lower ethanol concentration (6% v/v) became more susceptible to drying than nonacclimated ones, after rehydration they maintain their increased ability to grow in a synthetic wine. Acclimation at a higher ethanol concentration (10% v/v) produces several damages on the cell surface losing its ability to grow in a synthetic wine. CONCLUSIONS: In this work, we showed for the first time that sublethal alterations on bacterial surface induced by a pre-acclimation with a low ethanol concentration (6%), upon a freeze-drying process, result in a better bacterial adaptation to the stress conditions of wine-like medium, as well as to the preservation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the adaptation to ethanol of oenological strains and their effects on the preservation process has a strong impact on winemaking process and allows to define the most appropriate conditions to obtain malolactic starters cultures.

Structural and thermodynamic properties of water-membrane interphases: significance for peptide/membrane interactions.

Water appears as a common intermediary in the mechanisms of interaction of proteins and polypeptides with membranes of different lipid composition. In this review, how water modulates the interaction of peptides and proteins with lipid membranes is discussed by correlating the thermodynamic response and the structural changes of water at the membrane interphases. The thermodynamic properties of the lipid-protein interaction are governed by changes in the water activity of monolayers of different lipid composition according to the lateral surface pressure. In this context, different water populations can be characterized below and above the phase transition temperature in relation to the CH2 conformers' states in the acyl chains. According to water species present at the interphase, lipid membrane acts as a water state regulator, which determines the interfacial water domains in the surface. It is proposed that those domains are formed by the contact between lipids themselves and between lipids and the water phase, which are needed to trigger adsorption-insertion processes. The water domains are essential to maintain functional dynamical properties and are formed by water beyond the hydration shell of the lipid head groups. These confined water domains probably carries information in local units in relation to the lipid composition thus accounting for the link between lipidomics and aquaomics. The analysis of these results contributes to a new insight of the lipid bilayer as a non-autonomous, responsive (reactive) structure that correlates with the dynamical properties of a living system.

Virulence of Bacillus cereus: a multivariate analysis.

Biological activity and presence of DNA sequences related to virulence genes were studied in 21 strains of the Bacillus cereus group. The activity of spent culture supernatants and the effect of infection by vegetative bacterial cells were assessed on cultured human enterocytes (Caco-2 cells). The effect of extracellular factors on the detachment, necrosis and mitochondrial dehydrogenase activity of cultured human enterocytes was studied. Hemolytic activity on rabbit red blood cells was also evaluated and the effect of direct procaryotic-eucaryotic interactions was assessed in infection assays with vegetative bacterial cells. Concerning virulence genes, presence of the DNA sequences corresponding to the genes entS, entFM, nhe (A, B and C), sph, hbl (A, B, C and D), piplC and bceT was assessed by PCR. Ribopatterns were determined by an automated riboprinting analysis after digestion of the DNA with EcoRI. Principal component analysis and biplots were used to address the relationship between variables. Results showed a wide range of biological activities: decrease in mitochondrial dehydrogenase activity, necrosis, cell detachment and hemolytic activity. These effects were strain-dependent. Concerning the occurrence of the DNA sequences tested, different patterns were found. In addition, ribotyping showed that strains under study grouped into two main clusters. One of these clusters includes all the strains that were positive for all the DNA sequences tested. Positive and negative correlations between variables under study were evidenced. Interestingly, high detaching strains were positively correlated with the presence of the sequences entS, nheC and sph. Within gene complexes, high correlation was found between sequences of the hbl complex. In contrast, sequences of the nhe complex were not correlated. Some strains clustered together in the biplots. These strains were positive for all the DNA sequences tested and they were able to detach enterocytes upon infection. Our results highlight the multifactorial character of the virulence of the B. cereus group and show the correlation between ribopatterns, occurrence of toxin genes and biological activity of the strains under study.

Characterization of liposomes coated with S-layer proteins from lactobacilli.

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.

Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination.

Age at death determination based on aspartic acid racemization in dentin has been applied successfully in forensic odontology for several years now. An age-dependent accumulation of D-aspartic acid has also recently been demonstrated in bone osteocalcin, one of the most abundant noncollagenous proteins of the organic bone matrix. Evaluation of these initial data on in vivo racemization of aspartic acid in bone osteocalcin was taken a step further. After purification of osteocalcin from 53 skull bone specimens, the extent of aspartic acid racemization in this peptide was determined. The D-aspartic acid content of purified bone osteocalcin exhibited a very close relationship to age at death. This confirmed identification of bone osteocalcin as a permanent, 'aging' peptide of the organic bone matrix. Its D-aspartic acid content may be used as a measure of its age and hence that of the entire organism. The new biochemical approach to determination of age at death by analyzing bone is complex and demanding from a methodologic point of view, but appears to be superior in precision and reproducibility to most other methods applicable to bone.

Regulation of the human Gi alpha-2 gene promotor activity in embryonic chicken cardiomyocytes.

Increased expression of the inhibitory G protein Gi alpha-2 is assumed to contribute to desensitization of adenylyl cyclase in human heart failure. The mechanisms of upregulation involve increases in myocardial Gi alpha-2 protein, mRNA and gene transcriptional activity. To elucidate these mechanisms in more detail, the 5' flanking region of the human Gi alpha-2 gene (-1214/+115 bp) was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected in embryonic chick cardiomyocytes. CAT activity was measured 48 h after transfection. Unstimulated activity of the -1214/+115 bp construct was about 10-fold higher than activity of the basal CAT-construct (pGEMCAT). 5' deletion from -1214/+115 to -85/+115 bp upstream of the transcriptional start site increased, further stepwise deletions to 46/+115 gradually decreased promotor activity. Deletion from -46/+115 to -33/+115 bp completely abolished promotor activity. Stimulation of cardiomyocytes that had been transfected with the -1214/+115 CAT-construct with isoprenaline (10 microM), forskolin (10 microM), forskolin (10 microM) plus IBMX (10 microM) or dibutyryl-cAMP (1 mM) for 24 h induced an increase in CAT activity to 139 +/- 12% (n = 9), 211 +/- 18% (n = 12), 256 +/- 20% (n = 5) and 198 +/- 28% (n = 7) of unstimulated values, respectively. We conclude: 1) In chicken cardiomyocytes a sequence element of 52 bp between -85 and -33 bp is necessary to provide basal Gi alpha-2 promotor activity. 2) Elevation of cAMP has a stimulatory effect on the human Gi alpha-2 promotor, thereby offering a mechanism for beta-adrenoceptor-mediated increases in Gi alpha-2 in the heart.

Unusual sequence of immunoglobulin L-chain rearrangements in a gamma heavy chain disease patient.

Patients with gamma heavy chain disease (gamma-HCD) generally produce incomplete immunoglobulin (Ig) gamma-heavy chains (gamma-HCD protein) which cannot associate with light chains (IgL). In most patients Bence Jones proteins (BJP) are not observed. However, in the 61-year-old patient WIN we found gamma l-HCD proteins and lambda BJP in serum and urine. WIN gamma l-HCD protein does not carry the Ig Fd region, has a molecular weight of 33.5 kDa, and the seven N-terminal amino acid residues are not translated from any of the known immunoglobulin heavy chain (IgH) gene sequences. These residues are followed by the C gamma l-hinge region. In DNA from peripheral blood lymphocytes of patient WIN we found bands representing dominant rearrangements in one of the two alleles of the IgH, Ig kappa and Ig lambda locus. Taken together, the data from protein and DNA analysis strongly suggest, albeit do not formally prove, that one dominant B-cell clone which carries a rearranged and a non-rearranged allele of each Ig locus produces gamma-HCD protein and lambda BJP. The productive lambda-gene rearrangement in this clone thus has not been preceded by abortive rearrangements in both kappa-locus alleles. Lymphocytes with an unusual sequence of IgL-chain gene activation seem to be involved in the case of gamma-HCD described here.

[Gamma-1 heavy chain disease with the demonstration of Bence-Jones proteins]. / Gamma-1-Schwerkettenkrankheit mit Nachweis von Bence-Jones-Proteinen.

We describe a 61-year-old patient suffering from gamma-1-heavy-chain disease (gamma 1-HCD) associated with Bence-Jones-lambda proteinemia and proteinuria. The analysis of the patients gamma 1-HCD protein (WIN) shows a deletion of the complete Fd fragment. The N-terminal seven amino-acid residue does not resemble any of the known immunoglobulin-heavy-chain variable regions. Unexpectedly, in PBL-DNA and in DNA from EBV-immortalized cells we found in addition to the expected predominantly rearranged Ig-lambda-light-chain gene a predominant rearrangement of an Ig-kappa gene. These findings show that the gamma-1-heavy-chain disease of the patient involves a defective regulation of Ig-light-chain-gene activation as well.

[Immunoglobulin and T-cell receptor gene rearrangements in patients with autoimmune diseases]. / Immunglobulin- und T-Zellrezeptor-Genrearrangements bei Patienten mit Autoimmunkrankheiten.

Immunoglobulin-(Ig-) and T-cell-receptor-(TcR-)gene rearrangements were investigated in peripheral blood lymphocytes (PBL) of patients with various autoimmune disorders. In patients with SLE there was no predominant Ig- or TcR-gene rearrangement. This was also true in patients with a long disease duration and with excessive hypergammaglobulinemia. These results lead us to suggest that B cells are activated polyclonally in these patients. In those cases, where predominantly rearranged Ig- or TcR-genes were found, the autoimmune disorder was associated with a low-grade non-Hodgkin lymphoma (NHL). This coherence of B-cell malignancy and autoimmunity was only found in patients with cryoglobulinemia (KG), cold agglutinin disease (KA), and hemolytic anemia (AIHA).

[Unusual immunoglobulin gene rearrangements in patients with immunodeficiency]. / Ungewöhnliche Immunglobulin-Gen-Rearrangements bei Patienten mit Immundefekt.

In a 64-year-old patient with typical common variable immunodeficiency (CVID) DNA prepared from peripheral blood lymphocytes (PBL) showed a prominent immunoglobulin heavy chain (IgH) gene rearrangement; the immunoglobulin light chain (IgL) genes were found to be in the germline configuration. In contrast, a 13-year-old girl with a hitherto unidentified immunodeficiency showed polyclonal IgH gene rearrangements and a dominant IgL gene rearrangement. In both cases neither monoclonal B-lymphocytes nor monoclonal immunoglobulins were detectable. Our explanation for this unusual observation is that V-gene use in a given B-cell is not entirely random. This may be the consequence of a maturation arrest of B-cells.

Predominant immunoglobulin gene rearrangements in two patients with immunodeficiency: restricted use of V gene segments and DNA hypermethylation.

The majority of common variable immunodeficiencies (CVID) is caused by intrinsic B cell defects which impede distinct stages of B cell differentiation. B cell differentiation is accompanied by the rearrangement of immunoglobulin (Ig) genes. The first step in the rearrangement process is the assembly of IgH genes, and subsequently, IgL genes are rearranged. During B cell maturation, Ig genes are demethylated in a stepwise, locus-specific manner. Here, we examined the Ig gene rearrangements of four patients with classical CVID and of one child suffering from an unusual immunodeficiency associated with CD5+ B cell lymphocytosis. In one of the four adult patients with CVID, we observed a predominant type of VHDJH-gene rearrangement. In the child, different polyclonal VHDJH-gene rearrangements were found together with a predominant type of kappa light chain gene rearrangement. The rearranged kappa chain genes were methylated (as in the pre-B cell stage). These findings together with the cell phenotype analysis and the clinical course of the disease in the child suggests that in some patients with primary immunodeficiency a maturation arrest may occur in B cells leading to a predominant Ig V gene rearrangement.

IgD/lambda plasmocytoma with immunoglobulin kappa light-chain genes in the germ-line configuration.

Human immunoglobulin (Ig) genes are rearranged in an ordered sequence of events during B-cell differentiation: starting at the IgH locus, a productive VHDJH rearrangement leads to the expression of mu chains. Light-chain gene rearrangements have been found in pre-B cells which express mu chains. In these cells rearrangements of Ig kappa light-chain genes precede that of lambda genes. In an IgD/lambda-producing plasmocytoma, however, we found an apparent exception to this rule: the kappa genes were not rearranged. Together with the observation that roughly 90% of human IgD plasmocytomas produce lambda light-chain proteins, the finding reported here leads us to suggest that lambda light-chain genes are rearranged preferentially in IgD-producing plasma cells. Ig gene rearrangement, isotype switch, and the phenomenon of isotypic and allelic exclusion are discussed with special reference to our findings.

[Unusual rearrangements of immunoglobulin genes in a patient with IgD/lambda plasmacytoma]. / Ungewöhnliche Immunglobulin-Gen-Rearrangements bei einem Patienten mit IgD/lambda-Plasmozytom.

The mechanisms of Ig gene expression in B-lymphocytes are not yet fully understood. A hierarchy of Ig gene rearrangements has been suggested starting with Ig heavy chain genes followed by kappa and lambda light chain genes. According to this principle, B-cells with productive lambda light chain expression have their kappa genes either unproductively rearranged or deleted. By examining B-cells from patients with various lymphoproliferative diseases the developmental hierarchy of the Ig gene rearrangements could be confirmed with one notable exception: in a patient with IgD/lambda-plasmocytoma we found the kappa genes in germ line configuration. This finding points to an aberrant light chain gene expression in IgD-producing plasma cells.

Mebendazole 500 mg for single-dose treatment of nematode infestation.

Single 500 mg doses of mebendazole (Vermox; Janssen) were administered to 211 children aged 5 - 16 years in a rural area for treatment of single and mixed infestations with Ascaris lumbricoides, hookworm and Trichuris trichiura; the majority of subjects had low egg counts. Treatment was particularly effective against Ascaris, less effective against hookworm and promising against Trichuris. In most refractory cases egg output was considerably reduced. Treatment resulted in fewer Ascaris (95%) and hookworm (84%) ova reaching the environment. The results compare well with those obtained elsewhere in the world. One case of Strongyloides stercoralis infestation appeared after treatment. The drug was well tolerated and no side-effects were observed.