Lipopeptides of the N-terminus of Escherichia coli lipoprotein: synthesis, mitogenicity and properties in monolayer experiments

The N-terminal part of the lipoprotein from the outer membrane of Escherichia coli, tripalmitoyl-S-glyceryl-L-Cys-Ser and analogs with longer sequences, are polyclonal activators for B-lymphocytes. Triple-chain lipopeptides also constitute efficient low-molecular-weight carrier/adjuvant systems, which can be linked to antigens to yield immunogens for antibody production without further additives. This is the first report of monolayer experiments with chemically well defined, synthetic lipopeptide mitogens with the composition of the N-terminus of an important bacterial membrane protein. Various derivatives of the lipoprotein N-terminus were synthesized. These lipopeptides differed in the length of the peptide moiety, the number of fatty acid residues, and protective groups. In order to obtain the surface areas for the lipopeptides in isotherms and hysteresis isotherms, monolayer experiments with a computer-controlled film balance were performed. To get some information about the interaction of these compounds with typical membrane lipids mixed monolayers were formed from triple-chain lipopeptides with dipalmitoylphosphatidylcholine and cholesterol. A comparison of the mitogenic response of the compounds was made in an in vitro system with B-lymphocytes from Balb/c mice.     

All-Trans Retinoic Acid in Combination with Primaquine Clears Pneumocystis Infection

Pneumocystis pneumonia (PcP) develops in immunocompromised patients. Alveolar macrophages play a key role in the recognition, phagocytosis, and degradation of Pneumocystis, but their number is decreased in PcP. Our study of various inflammatory components during PcP found that myeloid-derived suppressor cells (MDSCs) accumulate in the lungs of mice and rats with Pneumocystis pneumonia (PcP). We hypothesized that treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, may effectively control Pneumocystis (Pc) infection by inducing MDSCs to differentiate to AMs. In rodent models of PcP, we found that 5 weeks of ATRA treatment reduced the number of MDSCs in the lungs and increased the number of AMs which cleared Pc infection. We also found that ATRA in combination with primaquine was as effective as the combination of trimethoprim and sulfamethaxazole for treatment of PcP and completely eliminated MDSCs and Pc organisms in the lungs in two weeks. No relapse of PcP was seen after three weeks of the ATRA-primaquine combination treatment. Prolonged survival of Pc-infected animals was also achieved by this regimen. This is the very first successful development of a therapeutic regimen for PcP that combines an immune modulator with an antibiotic, enabling the hosts to effectively defend the infection. Results of our study may serve as a model for development of novel therapies for other infections with MDSC accumulation.     

Safety assessment of potential lactic acid bacteria Bifidobacterium longum SPM1205 isolated from healthy Koreans

The safety assessment of Bifidobacterium longum SPM1205 isolated from healthy Koreans and this strain's inhibitory effects on fecal harmful enzymes of intestinal microflora were investigated. The overall safety of this strain was investigated during a feeding trial. Groups of SD rats were orally administered a test strain or commercial reference strain B. longum 1 x 10(9) CFU/kg body weight/day for four weeks. Throughout this time, their feed intake, water intake and live body weight were monitored. Fecal samples were periodically collected to test harmful enzyme activities of intestinal microflora. At the end of the four-week observation period, samples of blood, liver, spleen, kidney, and gut tissues were collected to determine for hematological parameters and histological differences. The results obtained in this experiment demonstrated that four weeks of consumption of this Bifidobacterium strain had no adverse effects on rat's general health status, blood biochemical parameters or histology. Therefore, it is likely to be safe for human use. Fecal harmful enzymes such as beta-glucosidase, beta-glucuronidase, tryptophanase and urease, were effectively inhibited during the administration of the B. longum SPM1205. These results suggested that this B. longum SPM 1205 could be used for humans as a probiotic strain.     

<Emphasis Type="Italic">Arcobacter acticola</Emphasis> sp. nov., isolated from seawater on the East Sea in South Korea

A Gram-stain-negative, facultative aerobic, non-flagellated, and rod-shaped bacterium, designated AR-13^T, was isolated from a seawater on the East Sea in South Korea, and subjected to a polyphasic taxonomic study. Strain AR-13^T grew optimally at 30°C, at pH 7.0–8.0 and in the presence of 0–0.5% (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain AR-13^T fell within the clade comprising the type strains of Arcobacter species, clustering coherently with the type strain of Arcobacter venerupis. Strain AR-13^T exhibited 16S rRNA gene sequence similarity values of 98.1% to the type strain of A. venerupis and of 93.2–96.9% to the type strains of the other Arcobacter species. Strain AR-13^T contained MK-6 as the only menaquinone and summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), C_16:0, C_18:1ω7c, and summed feature 2 (iso-C_16:1 I and/or C_14:0 3-OH) as the major fatty acids. The major polar lipids detected in strain AR-13^T were phosphatidylethanolamine, phosphatidylglycerol, and one unidentified aminophospholipid. The DNA G+C content was 28.3 mol% and its mean DNA-DNA relatedness value with the type strain of A. venerupis was 21%. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain AR-13^T is separated from recognized Arcobacter species. On the basis of the data presented, strain AR-13^T is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter acticola sp. nov. is proposed. The type strain is AR-13^T (=KCTC 52212^T =NBRC 112272^T).     

pH-Abhängigkeit der 13C-15N-kopplungskonstanten 15N-hochmarkierter aminosäuren,gewonnen aus algenmassenkulturenpH dependence of 13C-15N coupling constants of highly 15N-enriched amino acid isolated from mass cultivation of algae

The alga Ankistrodesmus braunii was grown with [¹⁵N]nitrate under the optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The ¹⁵N-labelled amino acids (¹⁵N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The ¹⁵N content of the analytically pure amino acids was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the ¹⁵N analysator. Using pulse Fourier transform ¹³C nuclear magnetic resonance, the pH dependence of the ¹³C-¹⁵N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J. Am. Chem. Soc. 86, 5564–5570) between the coupling constant and the product of the S-part of the ¹³C and ¹⁵N hybridization $\text{S}{}_{\mathrm{C}}\text{·}\text{S}{}_{\mathrm{N}}\text{= 80 · J (}{}^{13}\text{C}\text{-}{}^{15}\text{X}\text{)}$ fits best in acidic medium. The magnitude of the coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153–182). The recording of ¹³C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the ¹³C-¹⁵N coupling constants of the amino acids.     

Physicochemical and biological analysis of synthetic bacterial lipopeptides: validity of the concept of endotoxic conformation

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle.     

The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine

In diagnostic pathology and immunocytochemical research, immunohistochemical techniques using the streptavidin–biotin–peroxidase system have played an extremely valuable role. This system, based on the high affinity of streptavidin for biotin, may, however, provoke false positive results because of endogenous streptavidin-binding sites in human tissues. With the advent of the antigen retrieval procedure and signal amplification method, this problem can be serious enough to cause mistakes in interpreting immunohistochemical staining results. Therefore, we examined the distribution of endogenous biotin-like molecules in various human tissues and the influence of various antigen retrieval procedures with or without signal amplification using biotinylated tyramine to reveal these biotin-like activities. We observed that endogenous biotin-like molecules were present in a wide range of tissues, and their activity was markedly enhanced by employing antigen retrieval procedures or signal amplification. Furthermore, the extent to which the activity of endogenous biotin-like activities was enhanced depended on the kinds of antigen retrieval procedures and signal amplification employed. Pressure cooking and tyramine amplification with microwave heating showed the highest activities. These results show that the antigen retrieval procedures and signal amplification with tyramine can enhance the activity of endogenous biotin or biotin-like molecules as well as antigenicity, which can be a pitfall in the interpretation of immunohistochemical data.     

The Chronological Characteristics of SOD1 Activity and Inflammatory Response in the Hippocampi of STZ-Induced Type 1 Diabetic Rats

Because it appears that oxidative stress and inflammation are implicated with disease pathogenesis in the diabetic brain, many researchers have used streptozotocin (STZ)-induced diabetic animals to study superoxide production and the effects of superoxide scavengers like Cu,Zn-superoxide dismutase (SOD1). However, many studies have been conducted without considering temporal changes after STZ injection. Interestingly, though SOD activities were not significantly different among the groups, SOD1 and 4-hydroxy-2-nonenal (4-HNE) immunoreactivities were significantly enhanced at 3 weeks after an STZ injection (STZ3w) versus only marginal levels in sham controls, whereas microglial activity was remarkably reduced in injected rats at this time. However, SOD1 immunoreactivity and microglial activities were only at the sham level at STZ4w. The present study provides important information concerning cell damage by ROS generated by STZ. Microglial response was found to be inactivated at STZ3w and neuronal cells (NeuN) showed a non-significant tendency to be reduced in number at STZ4w except in the dentate gyrus. We speculated that the above oxidative stress-related events should be accomplished at STZ3w in the brains of STZ-induced diabetes animal models. Therefore, the aim of the present study was to investigate chronological changes in SOD1 immunoreactivity associated with lipid peroxidation and inflammatory responses in the hippocampi of STZ-induced type I diabetic rats.