Quantitative aspects of the feeder cell phenomenon: mechanistic implications

It has been proposed that feeder cells function by supplying lymphocytes with the amino acid cysteine (a thiol compound). The results presented here indicate that thiols are the critical element of the feeder cell phenomenon. Specifically, we noted that the rank of thiol production by four different feeder cell lines corresponds to their relative abilities to support a lymphocyte cell line, CTLL-2. In addition, increasing thiol production by the feeder cells with lipopolysaccharide increased their support of CTLL-2 cells and decreasing it with homocysteate decreased support of CTLL-2 cells. However, it was also noted that substantial (up to 79% maximal) support of CTLL-2 growth was provided by feeder cell concentrations which could not produce detectable levels of free thiols. This prompted us to propose an alternative mechanism for the feeder effect which would explain these apparently paradoxical findings.     

Evaluation of nitrite production by human monocyte-derived macrophages.

Reactive nitrogen intermediates are important in the anti-tumor and anti-microbial activities of rodent macrophages, but it is not known whether this is the case for human macrophages. In the present study, nitrite concentrations in vitro were used as an indicator of reactive nitrogen intermediate production by mouse, rat, and human macrophages. Human macrophages derived by culturing peripheral blood monocytes did not consistently produce detectable nitrite levels in response to any stimulus examined. Human macrophages were viable and metabolically active as indicated by the MTT assay, and their respiratory burst response to phorbol myristate acetate was increased following incubation with Interferon-gamma, as expected for typical macrophages. In contrast, rat or mouse peritoneal macrophages produced nitrite concentrations of approximately 20-100 microM in response to lipopolysaccharide, Interferon-gamma, or both. These results demonstrate substantial differences in the production of nitrites by rodent and human macrophages. Because of the heterogeneity among macrophage populations, these findings may not be applicable to all human macrophage populations, but they suggest a need for caution in extrapolating from rodent studies regarding the role of reactive nitrogen intermediates in anti-tumor or anti-microbial functions of human macrophages.     

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.     

Effect of exposure to soil on potency and spore viability of Bacillus thuringiensis

Ten-gram samples of a clay loam soil were inoculated with Bacillus thuringiensis var. galleriae (H-serotype V) and held at 25°C. Periodically the spores and δ endotoxin protein crystals of B. thuringiensis were extracted from soil samples. Numbers of viable spores were estimated by plate counts and pathogenicity determined by bioassay with larvae of Galleria mellonella. During 135 days, the number of viable spores fell slowly to 24% of the initial numbers, while pathogenicity fell rapidly to <1%, which suggests that the crystals were degraded far more rapidly than spores. Natural soil bacteria increased in numbers during the same period.     

Multiple cannulation of the primate superficial lateral coccygeal vein.

The superficial lateral coccygeal veins of Macaca fascicularis were exposed surgically and cannulated with polyethylene tubing. The cannula was used for administering continuous infusions or obtaining multiple blood samples, and it was removed 12--18 hours after insertion.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Impaired Eye Region Search Accuracy in Children with Autistic Spectrum Disorders

To explore mechanisms underlying reduced fixation of eyes in autism, children with Autistic Spectrum Disorders (ASD) and typically developing children were tested in five visual search experiments: simple color feature; color-shape conjunction; face in non-face objects; mouth region; and eye region. No group differences were found for reaction time profile shapes in any of the five experiments, suggesting intact basic search mechanics in children with ASD. Contrary to early reports in the literature, but consistent with other more recent findings, we observed no superiority for conjunction search in children with ASD. Importantly, children with ASD did show reduced accuracy for eye region search (p = .005), suggesting that eyes contribute less to high-level face representations in ASD or that there is an eye region-specific disruption to attentional processes engaged by search in ASD.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Induced fit in guanidino kinases--comparison of substrate-free and transition state analog structures of arginine kinase

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 A with a final R of 22.3% (R(free) = 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.     

Molecular and Biochemical Diagnosis of Esterase-Mediated Pyrethroid Resistance in a Mexican Strain of Boophilus microplus (Acari: Ixodidae)

We examined pyrethroid resistant Mexican strains of Boophilus microplus using biochemical and molecular tests to determine the mechanisms conferring resistance. Permethrin hydrolysis assays and esterase activity gels indicated enhanced esterase-mediated metabolic detoxification in the Cz strain, while one other pyrethroid resistant strain, SF, and two pyrethroid susceptible strains had lower levels of permethrin hydrolysis. Results from assays using a PCR-based test to detect a pyrethroid target site resistance-associated mutation in the tick sodium channel gene found only low levels of mutations in the Cz strain, while the SF strain had a high level of the mutated sodium channel alleles. A specific esterase, designated CzEst9, believed to be responsible for the esterase-mediated pyrethroid resistance in the Cz strain was purified, and the gene encoding CzEst9 cloned. This revised version was published online in July 2006 with corrections to the Cover Date.