Isolation of linoleic acid as an estrogenic compound from the fruits of Vitex agnus-castus L. (chaste-berry).

A methanol extract of chaste-tree berry (Vitex agnus-castus L.) was tested for its ability to displace radiolabeled estradiol from the binding site of estrogen receptors alpha (ERalpha) and beta (ERbeta). The extract at 46 +/- 3 microg/ml displaced 50% of estradiol from ERalpha and 64 +/- 4 microg/ml from ERbeta. Treatment of the ER+ hormone-dependent T47D:A18 breast cancer cell line with the extract induced up-regulation of ERbeta mRNA. Progesterone receptor (PR) mRNA was upregulated in the Ishikawa endometrial cancer cell line. However, chaste-tree berry extract did not induce estrogen-dependent alkaline phosphatase (AP) activity in Ishikawa cells. Bioassay-guided isolation, utilizing ER binding as a monitor, resulted in the isolation of linoleic acid as one possible estrogenic component of the extract. The use of pulsed ultrafiltration liquid chromatography-mass spectrometry, which is an affinity-based screening technique, also identified linoleic acid as an ER ligand based on its selective affinity, molecular weight, and retention time. Linoleic acid also stimulated mRNA ERbeta expression in T47D:A18 cells, PR expression in Ishikawa cells, but not AP activity in Ishikawa cells. These data suggest that linoleic acid from the fruits of Vitex agnus-castus can bind to estrogen receptors and induce certain estrogen inducible genes.

[2+2] versus [3+2] addition of metal oxides across C=C double bonds: toward an understanding of the surprising chemo- and periselectivity of transition-metal-oxide additions to ketene.

The peri-, chemo-, stereo-, and regioselectivity of the addition of the transition-metal oxides OsO4 and LReO3 (L = O-, H3PN, Me, Cp) to ketene were systematically investigated using density-functional methods. While metal-oxide additions to ethylene have recently been reported to follow a [3+2] mechanism only, the calculations reveal a strong influence of the metal on the periselectivity of the ketene addition: OsO4 again prefers a [3+2] pathway across the C=C moiety whereas, for the rhenium oxides LReO3, the [2+2] barriers are lowest. Furthermore, a divergent chemoselectivity arising from the ligand L was found: ReO4- and (H3PN)ReO3 add across the C=O bond while MeReO3 and CpReO3 favor the addition across the C=C moiety. The calculated energy profile for the MeReO3 additions differs from the CpReO3 energy profile by up to 45 kcal/mol due to the stereoelectronic flexibility of the Cp ligand adopting eta5, eta3, and eta1 bonding modes. The selectivity of the cycloadditions was rationalized by the analysis of donor-acceptor interactions in the transition states. In contrast, metal-oxide additions to diphenylketene probably follow a different mechanism: We give theoretical evidence for a zwitterionic intermediate that is formed by nucleophilic attack at the carbonyl moiety and undergoes a subsequent cyclization yielding the thermodynamically favored product. This two-step pathway is in agreement with the results of recent experimental work.

Thermal, catalytic, regiospecific functionalization of alkanes

The formation of a single product from terminal functionalization of linear alkanes from a transition metal-catalyzed reaction is reported. The rhodium complex Cp*Rh(eta(4)-C(6)Me(6)) (Cp*, C(5)Me(5); Me, methyl) catalyzes the high-yield formation of linear alkylboranes from commercially available borane reagents under thermal conditions. These reactions now allow catalytic, regiospecific functionalization of alkanes under thermal conditions. The organoborane products are among the most versatile synthetic intermediates in chemistry and serve as convenient precursors to alcohols, amines, and other common classes of functionalized molecules.

Identification of a novel core type in Salmonella lipopolysaccharide. Complete structural analysis of the core region of the lipopolysaccharide from Salmonella enterica sv. Arizonae O62.

For the first time, the complete structure of a lipopolysaccharide (LPS) core region from Salmonella enterica has been identified that is different from the Ra core type generally thought to be present in all Salmonella LPS. The LPSs from two rough mutants and the smooth form of S. enterica sv. Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy. In the novel core type, the terminal D-GlcNAc residue present in the Ra core type, is replaced by a D-Glc residue. The O-specific polysaccharide is alpha1-->4-linked to the second distal Glc residue of the core. Furthermore, phosphoryl substituents attached to O-4 of L-glycero-D-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II). [structure: see text] Abbreviations in Structure I are as follows: Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide. The presence of this novel core type in LPS of S. enterica should be taken into account in the development of a general antibody-based diagnostic system for Salmonella.

Culture and biological activity of Propionibacterium acnes.

Administration of killed Propionibacterium acnes to experimental animals leads to the development of hypersensitivity to the lethal and cytokine-inducing effects of endotoxin. This sensitizing property of P. acnes is not always expressed by different bacterial preparations. Its expression depends very much on the conditions employed for the cultivation of this microorganism. The present study investigates which culturing conditions result in P. acnes preparations with optimal sensitizing properties. The composition of the medium, the culturing time and temperature as well as the type of cultivation (in minifermentor or stationary culture) were all varied for this purpose. The resulting bacterial preparations were killed at 65 degrees C for 1 h and tested for sensitizing activity. The results show that stationary cultures of P. acnes grown at 37 degrees C for 4 to 5 days in the appropriate medium produce biologically active preparations with satisfactory sensitizing activity.

Protection against bacterial infection by urea extracts from Salmonella.

Immunization assays were performed in NMRI mice using urea extracts from both the S-form and an R-mutant of S. typhimurium. Our results show that, apart from providing protection against infection, both vaccines induced a DTH (delayed type hypersensitivity) reaction against the antigen extract. The strength of the DTH reaction depended on the type of test antigen used, the urea extracts proving to be superior to extracts obtained by ultrasonication. With the urea extract, all animals responded in the foot pad test. Both parameters of the immune response, protection against infection and the strength of DTH, could be further enhanced by a lipopeptide adjuvant, which was effective both in mixture and conjugate form; the humoral immune response was not enhanced. Thus, urea extract vaccines from S. typhimurium induce a cell-mediated response which can be further enhanced by lipopeptide adjuvants.

The sialic acid-containing lipopolysaccharides of Salmonella djakarta and Salmonella isaszeg (serogroup O: 48): chemical characterization and reactivity with a sialic acid-binding lectin from Cepaea hortensis.

Lipopolysaccharides (LPS) of Salmonella djakarta and Salmonella isaszeg, as well as of a spontaneous R-mutant of S. djakarta were investigated as to their content in neuraminic acid (Neu) and its individual linkage. The two Salmonella serovars both belong to the O:48 serogroup of Salmonella, but to two different subgroups. LPS of both S-forms contained high amounts of Neu, although in different quantities, whereas the R-form was completely devoid of it. Methylation analysis indicated that Neu is exclusively terminally linked in S. djakarta whereas both terminal and 4-linked Neu were recognized in S. isaszeg. Although terminally linked, a sialidase from Arthrobacter ureafaciens was unable to split Neu even after prolonged incubation from both S-type LPSs. When LPS was first treated by mild alkali, however, the total amount of Neu from S. djakarta LPS and about 50% from that of LPS of S.isaszeg could be removed. In contrast, alkali-treated LPS, but also the non-treated one, proved to be effective inhibitors for a sialic acid-binding lectin from Cepaea hortensis. The resistance of terminal Neu towards sialidase may be due to the presence of an O-acetyl group which would be removed during the methylation analysis but would, especially when linked to C-4, not interfere with the reactivity of the lectin.

Influence of the glucose concentration on the yield of biomass and lipopolysaccharide in Salmonella cultures.

Two Salmonella S-forms and two R-mutants were cultivated in complex medium supplemented with different amounts (0.5-3%) glucose. Cultivation was performed batchwise in a fermentor under aerobic conditions. With all strains investigated, the yield of bacterial mass increased with increasing concentration of glucose. In the case of three strains, the % LPS content of bacteria also increased, thus achieving the aim of this investigation. The synthesis of bacterial mass and LPS did not proceed in parallel and differed from strain to strain. At optimal glucose concentration, the yield of LPS could be increased up to 250%. The chemical composition of the LPS was independent of the glucose concentration. The individual strains exhibited an identical composition with regard to lipid A and polysaccharide when cultured at different glucose concentrations. The uniformity of the molecular distribution of LPS could also be confirmed by SDS-polyacrylamide gel electrophoresis. In the S-form LPSs, also the proportion of the unsubstituted R-form LPS was not affected by the glucose concentration in the culture medium. The present results demonstrate that optimisation of the cultivation conditions with respect to the glucose concentration of the medium would be of advantage especially for Salmonella strains that are cultivated frequently.

The influence of low growth temperature on the amount of free R lipopolysaccharide, on the expression of R-core determinants and on O-chain lengths in Salmonella S forms.

Ten Salmonella strains belonging to five serological groups were cultivated both at 37 degrees C and close to their individual temperature minima at 12-14 degrees C and the composition of their cell wall lipopolysaccharides (LPS) was compared. When grown at low temperature, the proportion of unsubstituted (free) R-LPS in the total LPS moiety increased significantly in 6 strains, whereas in the other strains, no change or even a slight decrease in the R-LPS proportion was observed as judged from the analyses by SDS-PAGE. In the immunoblot, the R-LPSs from 9 out of 10 strains showed a modified reactivity against a set of specific Salmonella R antisera (anti-Ra, anti-Rb1, anti-Rb2, anti-Rc). In most cases, the decrease in Rb1 reactivity was paralleled by an increase in Rb2 reactivity and also by an increase in the total amount of free R-LPS. The electrophoretic mobility of free R-LPS was changed in 7 out of 10 strains, although the changes were not unidirectional. All changes occurred only in the range of the Ra-Rb1 chemotypes and no significant correlation to the serological grouping of the strains was evident. When grown at low temperature, the average number of O-repeating units was reduced in the majority of cases and in some cases, also the banding profile in SDS-PAGE in the S-LPS region was modified. The fatty acid spectra showed some changes which were in accordance with previous results, namely a decrease in the content of C-12:0 and C-16:0 and an increase in that of C-14:0 and C-16:1. The different influences of the growth temperature on the LPS biosynthesis of different Salmonella strains may be a result of the genetic diversity of this group of microorganisms.

The structure of the O-specific polysaccharide of Salmonella arizonae O62.

The O-specific polysaccharide was liberated by mild acid hydrolysis of the lipopolysaccharide (LPS) isolated from S. arizonae O62 by phenol-water extraction. The branched hexasaccharide repeating-unit of the O-specific chain of the O62 LPS contained L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galacturonic acid in molar ratios of 4:1:1. On the basis of methylation analysis, 1H and 13C NMR spectroscopy, including 2D shift-correlated (COSY) and 1D NOE spectroscopy, the following structure for the repeating unit of the O-specific polysaccharide was established: [formula: see text]

[Lipopeptides as natural adjuvants for vaccines from Gram-negative bacteria]. / Lipopeptide als natürliche Adjuvantien für Impfstoffe aus Gram-negativen Bakterien.

Bacterial cell wall components such as lipopolysaccharide, a variety of membrane proteins, murein, and lipoprotein can act as immunoadjuvants for bacterial vaccines, thus enhancing protection from bacterial infections. Synthetically prepared N-terminal parts of the lipoprotein from Enterobacteria carrying three fatty acid residues or lipopeptide analogs containing one to four aminoacids bound to S-glycerylcysteine act as potent immunoadjuvants in vivo in combination with or covalently linked to antigens. Here we demonstrate that the supplementation of Salmonella vaccines with these synthetic lipopeptides significantly enhances their vaccine efficiency in mice. Variations in the native lipopeptide structure regarding chain length and amino acid sequence of the peptide moiety, as well as modifications of the lipoamino acid, lead to reduction or even complete loss of the adjuvant activity. The immunoadjuvant properties of the lipopeptides as described here are mediated by an enhancement of the humoral immune response.

Immunoblot analysis of the R-form lipopolysaccharide from Salmonella S forms.

Lipopolysaccharide (LPS) preparations from Salmonella S-form bacteria contain, in addition to S-form LPS, variable amounts of R-form material, lacking the O-specific polysaccharide. In the present study, we investigated the R-form LPS present in 22 LPS preparations derived from different Salmonella S-form strains belonging to 8 serological groups. The R-form part of the LPSs was separated by SDS-polyacrylamide gel electrophoresis and its antigen specificity analyzed by subsequent immunoblotting using Salmonella R-antisera (anti-Ra, -Rb1, -Rb2, -Rc). The results show that different R-determinants were present in one and the same LPS preparation. The pattern of reaction varied considerably among individual strains. This variation was also present among strains of the same serogroup. Approximately one half of the R-form LPSs exhibited a weak or no reaction with Ra antiserum. In contrast, almost all R-form LPSs showed a significant reaction with Rb1 antiserum. The electrophoretic mobility of the R-form material was very similar, exhibiting only minor differences among the different LPS preparations. In most cases, migration was similar to that of authentic Salmonella Ra-LPS; in some cases, the migration was somewhat faster resembling that of Rb1-LPS.

Structural differences in the outer core region of lipopolysaccharides derived from members of the genus Salmonella.

Spontaneous and P22-resistant rough mutants, respectively, selected from Salmonella IV (18: z36, z38:-) and S. djakarta (48: z4, z24:-), appeared to lack the epitope recognized by the T6 monoclonal antibody which had been previously shown to correspond to the terminal alpha-1,2-linked N-acetyl-D-glucosamine residue of the Salmonella lipopolysaccharide (LPS) Ra core. LPSs and core oligosaccharides were therefore prepared from these two rough mutants and analysed by chemical and serological methods. Sugar analyses as well as methylation and 13C-NMR studies indicated that rough mutants derived from these two serotypes indeed possessed outer core structures differing from those of the well-characterized Salmonella Ra core. Serological data corroborated the chemical findings. Proposed structures of the outer core regions of these two R-types are presented and the significance of the findings is discussed.

Lack of the alpha-1,2-linked N-acetyl-D-glucosamine epitope in the outer core structures of lipopolysaccharides from certain O serogroups and subspecies of Salmonella enterica.

A total of 176 strains of Salmonella enterica representing 116 serotypes were tested for the presence of the T6 epitope of the alpha-1,2-linked N-acetyl-D-glucosamine residue by reaction with a murine monoclonal antibody T6 specific for this structure in the Salmonella Ra core lipopolysaccharide (LPS). All 20 serotypes (70 strains) belonging to serogroups A to E were positive for the T6 epitope while 29% of the 96 serotypes (106 strains) belonging to O serogroups F to 67 were negative; 12 serotypes (12 strains) of subspecies IIIb Salmonella were positive for the T6 epitope, but 10 serotypes (11 strains) of subspecies IIIa Salmonella were found to lack this epitope. In T6-positive strains, the epitope was accessible to antibody binding in both the unsubstituted free rough core LPS and in the rough core LPS substituted with a few repeating units of O side chains. The presence or absence of the T6 epitope in Salmonella strains was not affected by culture conditions, the source of the isolate, the age of the culture or the presence of fimbriae antigens.

A murine monoclonal antibody that recognizes a genus-specific epitope in the Salmonella lipopolysaccharide outer core.

A murine monoclonal antibody 105 made from spleen cells of a mouse immunized with a mixture of common Salmonella serotypes reacted specifically with salmonellae from the most frequently encountered O serogroups of A (O:2) to E (O:3), and with strains from the less common O serogroups that represent the subspecies I, II, IIIb, IV, V and VI. Specificity for Salmonella was demonstrated by the lack of reactivity of monoclonal antibody 105 with any of the 30 other different species of Gram-positive and Gram-negative bacteria tested including 16 species in the family of Enterobacteriaceae. Studies to elucidate its binding epitope have shown that it reacts with the three distal sugar residues joined through specific anomeric linkages as present only in the Salmonella lipopolysaccharide outer core, which explains its specificity for the Salmonella. The failure of monoclonal antibody 105 to react with a subspecies IIIa Salmonella suggested a different outer core structure in this strain of Salmonella and also that monoclonal antibodies to the outer core of Salmonella lipopolysaccharide should be useful in the molecular analysis of their diversity.

Smooth lipopolysaccharide of Salmonella adelaide has an atypical Salmonella Ra core.

Two wild isolates as well as two laboratory strains of Salmonella adelaide obtained from different geographical areas failed to react with a monoclonal antibody directed against the terminal alpha-1,2-linked N-acetylglucosamine residue of the outer core of Salmonella lipopolysaccharide (LPS). This finding was confirmed by the lack of reactivities of Salmonella Ra LPS with S. adelaide antiserum or of S. adelaide R oligosaccharide with Salmonella Ra serum. Furthermore, S. adelaide proved to be resistant to lysis by phage FO1, which binds to a receptor also believed to involve the terminal alpha-1,2-linked N-acetylglucosamine of the Salmonella R oligosaccharide. These results suggest that the outer core structure of S. adelaide LPS may be different from that of S. typhimurium and other Salmonella strains.

Enhancement of protection against Salmonella infection in mice mediated by a synthetic lipopeptide analogue of bacterial lipoprotein in S. typhimurium vaccines.

Vaccines consisting of acetone-killed Salmonella typhimurium were supplemented with a synthetically prepared lipopeptide derivative of bacterial lipoprotein, Pam3Cys-Ser-Ser-Asn-Ala. NMRI mice were immunized with these vaccines, receiving two intraperitoneal injections and were challenged intraperitoneally with graded doses of S. typhimurium C5. The protective capacity of the supplemented vaccines was compared with that of the unsupplemented bacterial vaccine, and with the effectiveness of the supplementing component alone. The LD50 served as a criterion for protective capacity. The results showed that 90% of the S. typhimurium S-form vaccine could be replaced by the adjuvant lipopeptide without a recognizable decrease in protective immunizing capacity. A similar but less pronounced enhancement of protection was obtained with a R-mutant vaccine supplemented with the lipopeptide; by supplementing the standard vaccine dose with lipopeptide an increase in protection was also achieved. Lipopeptide alone was not effective in protecting mice from infection with S. typhimurium.

Temperature-dependent incorporation of 4-amino-L-arabinose in lipid A of distinct gram-negative bacteria.

The presence and the relative amount of 4-amino-L-arabinose in lipopolysaccharides of members of the Enterobacteriaceae family and in a single strain of Chromobacterium violaceum has been studied with regard to growth-temperature dependent variations. Changes in the presence and the amount of 4-amino-L-arabinose (4-AA) were observed in almost all cases, but the variations observed were not consistent among different species. While Salmonella minnesota and Proteus mirabilis showed higher levels of incorporation at higher temperatures, the S- and R-forms of Yersinia enterocolitica showed the opposite effect, i.e. only marginal incorporation by growth at 10 degrees C. Chromobacterium violaceum, however, showed no significant alteration in the 4-amino-L-arabinose content when growth either at 14 or at 37 degrees C. DOC-PAGE pattern of isolated lipopolysaccharides showed characteristic profiles indicating that the O-chain-synthesis of distinct Enterobacteriaceae is also differently influenced by changes in growth temperature.