Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo.

The survival of Salmonella montevideo during serum treatment depends on the presence of an O antigen (O-Ag) associated with the lipopolysaccharide molecule. In this organism, the O antigen is a polysaccharide composed of 0 to more than 55 subunits, each containing 4 mannose residues together with glucose and n-acetylglucosamine. We used a mutant strain of S. montevideo that requires exogenous mannose for the synthesis of O-Ag. Lipopolysaccharide (LPS) was prepared from these cells grown under three different conditions where the availability of exogenous mannose was regulated such that the average number of O-Ag units per LPS molecule, the percentage of LPS molecules bearing long O-Ag side chains, and the percentage of lipid A cores bearing O-Ag were all varied. These changes in LPS profiles were monitored on sodium dodecyl sulfate-polyacrylamide gels, and cells with different LPS profiles were tested for their ability to survive treatment with pooled normal human serum. Survival in serum was associated with LPS that contained an average of 4 to 5 O-Ag units per LPS molecule, and 20 to 23% of the LPS molecules had more than 14 O-Ag units per LPS molecule. Serum survival was less clearly associated with the percentage of lipid A cores covered with O-Ag. We propose, based on these data and on previous work, that the O-Ag polysaccharide provides the cell protection from serum killing by sterically hindering access of the C5b-9 complex to the outer membrane and that a critical density of long O-Ag polysaccharide is necessary to provide protection.     

Properties and Characteristics of a Bacteriocin from Serratia marcescens

A strain of Serratia marcescens was found to produce a bacteriocin that inhibits the growth of certain Escherichia coli strains. This inhibition was bacteriocidal rather than bacteriostatic and was not caused by a bacteriophage. Whereas the bacteriocin was inactive on the 7 Serratia strains tested, it killed 11 of the 20 E. coli strains tested for sensitivity. A relationship of the bacteriocin to a possible colicin cannot as yet be excluded, although E. coli mutants resistant to 1 or 2 of 15 different colicins remained sensitive to the bacteriocin. The bacteriocidal effect by the bacteriocin could be interrupted in a substantial fraction of the treated cell population by the addition of trypsin. The synthesis of the bacteriocin was inducible by ultraviolet light or by starvation for thymidine. Both procedures led to a similar increase in maximum bacteriocin titer relative to noninduced cultures.     

A graph theoretic approach to the development of minimal phylogenetic trees

Summary The problem of determining the minimal phylogenetic tree is discussed in relation to graph theory. It is shown that this problem is an example of the Steiner problem in graphs which is to connect a set of points by a minimal length network where new points can be added. There is no reported method of solving realistically-sized Steiner problems in reasonable computing time. A heuristic method of approaching the phylogenetic problem is presented, together with a worked example with 7 mammalian cytochrome c sequences. It is shown in this case that the method develops a phylogenetic tree that has the smallest possible number of amino acid replacements. The potential and limitations of the method are discussed. It is stressed that objective methods must be used for comparing different trees. In particular it should be determined how close a given tree is to a mathematically determined lower bound. A theorem is proved which is used to establish a lower bound on the length of any tree and if a tree is found with a length equal to the lower bound, then no shorter tree can exist.     

Leaf manganese concentrations as a tool to assess belowground plant functioning in phosphorus-impoverished environments

Plant and Soil - Root-released carboxylates enhance the availability of manganese (Mn), which enters roots through transporters with low substrate specificity. Leaf Mn concentration ([Mn]) has been...     

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

Methodology/Principal Findings

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

Conclusions/Significance

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.     

Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.