Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Thymus-independent immunogenicity in vitro of the divalent antigen DNP-polyethylene oxide

Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.     

On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

Triamcinolone acetonide inhibits lymphocyte differentiation in B cells decorated with artificial antigen receptors

We examined the effects of triamcinolone acetonide (TA) on T cell independent antigen-induced differentiation of human B cells. Purified human B cells artificially decorated with palmitate-conjugated monoclonal IgA antibody specific for 2,4-dinitrophenyl differentiated polyclonally when challenged with optimum concentrations of dinitrophenyl-derivatized polymerized flagellin. This B cell response was reduced by the synthetic corticosteroid TA at a concentration of 10(-6) M. This suggests that TA can inhibit in vitro B lymphocyte differentiation independent of T cells.     

Photobleaching recovery studies of membrane events accompanying lectin stimulation of rabbit lymphocytes.

Fluorescence photobleaching recovery methods reveal marked changes in lateral mobilities of rabbit lymphocyte membrane components during the course of stimulation with succinyl concanavalin A (S Con A). The diffusion constant of S Con A receptors on T lymphocytes falls from 1.6×10^?10 cm²/sec to 6.5×10^?11 cm²/sec within 4 hr after stimulation, remains constant for 14 hr, and returns to its former value. The mobility of B cell receptors similarly falls from 1.4×10^?10 cm²/sec to 5.5×10^?11 cm²/sec but regains its unstimulated value much more slowly. In contrast, a fluorescent phospholipid analog shows constant mobilities of 1.9×10^?8 cm²/sec and 1.5×10^?8 cm²/sec in T and B cells, respectively, throughout the experiment.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.      

A review of the subspecies status of the Icelandic Purple Sandpiper Calidris maritima littoralis

The Icelandic Purple Sandpiper Calidris maritima littoralis (C.L. Brehm, 1831) represents one member of a poorly understood subspecies complex. Currently, differences in size define two other subspecies: Calidris maritima belcheri Engelmoer & Roselaar, 1998, which breeds in north‐eastern Canada along the Hudson Bay and James Bay, and Calidris maritima maritima (Brunnich, 1764), which breeds along the Arctic coasts elsewhere in northern Canada, Greenland, Svalbard, Scotland, and Fennoscandia, to northern central Siberia. There are large size differences amongst populations of C. m. maritima, however. As an Arctic/Alpine breeding bird, C. m. littoralis could provide an interesting perspective on the evolutionary changes following a northwards expansion of a species after glacial retreat. Considering the extent of the ice sheet in the northern hemisphere during the last glaciation, and the short period of time since it ended, the correct attribution of subspecies status for C. m. maritima may reflect either rapid diversification from a single population or ancestral splits of distinct evolutionary lineages that survived in isolation at southern latitudes. We applied morphometric subspecies criteria, diagnosability by Amadon's rule, and genetic analysis of five nuclear introns, and the mitochondrial DNA markers cytochrome oxidase c subunit I (COI) and NADH dehydrogenase subunit 2 (ND2), to geographically separate breeding populations in order to examine the subspecies status of the Icelandic population. The results do not provide support for the subspecies status of the Icelandic population because the nominate and Icelandic subspecies fail to uphold Amadon's rule, and genetic analyses indicate that the study populations derive from a single shared refugium. © 2015 The Linnean Society of London     

Insulin Receptors and Downstream Substrates Associate with Membrane Microdomains after Treatment with Insulin or Chromium(III) Picolinate

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)₃). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)₃. These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)₃ but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)₃ did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)₃ likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)₃ involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)₃ are due to changes in membrane lipid order rather than to direct interactions with IR.     

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.