Photobactin: a catechol siderophore produced by Photorhabdus luminescens,an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1 nematodes

The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction. Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD. The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity. This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity. The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae. Other putative siderophore biosynthetic and transport genes flanking phbH were characterized. The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid [4-(2,3-dihydroxybenzoylamino)-butyl]-amide, and it was given the generic name photobactin. Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver. These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction.     

Analysis of immunohistochemical label of Fos protein in the suprachiasmatic nucleus: comparison of different methods of quantification

The induction of the proto-oncogene c-fos, and its phosphoprotein product Fos, has been extensively used to study the effects of light on the circadian pacemaker in the suprachiasmatic nucleus (SCN). Experimental approaches to the quantification of Fos induction have mainly been based on immunohistochemistry and subsequent measure of Fos immunoreactivity (IR) in sections of the SCN. In this study, the authors compare several methods of quantification using optical density image analysis or counts of Fos-IR labeled cells. To assess whether optical density measures using image analysis reflect the amount of Fos in brain tissue, the authors developed standards of known concentrations of Fos protein in an agar matrix. The agar standards were sectioned and treated simultaneously with sections of the SCN from animals exposed to different levels of irradiance. Optical density was found to be proportional to the quantity of Fos in the sections, indicating that this measure accurately reflects relative levels of Fos protein induction. Quantification by optical density analysis allows an objective measure in which the various parameters, conditions of illumination, and threshold can be maintained constant throughout the analysis. Counting cells by visual observation is more subjective because threshold values cannot be precisely defined and can vary according to the observer, illumination, degree of label, and other factors. In addition, cell counts involving direct visual observation, automated cell counts, or stereological methods do not take into account the difference in the density of label between cells, thus giving equal weight to lightly or densely stained cells. These measures are more or less weakly correlated with measures of optical density and thus do not accurately reflect the amount of bound Fos protein in the tissue sections. In contrast, labeled surface area as measured by image analysis shows a linear relationship with optical density. The main outcome of this study is that computer-assisted image analysis provides an accurate and rapid method to determine the relative amount of Fos protein in the SCN and the effects of light on intracellular signaling mechanisms involved in the circadian clock.     

Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs

Delayed (or incomplete) ossification of developing fetal bones and wavy ribs are some of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. Although they tend to be regarded as minor effects, they can be quite sensitive and consequently may influence the study lowest-observed-adverse-effect levels (LOAELs), and thus, impact classification, labeling, and risk assessment. In this study, we review the underlying mechanisms of these skeletal variations, evaluate different scenarios in which they have been observed, offer guidance for their interpretation, and comment on their use for risk assessment. Both minor delays in ossification and wavy ribs seem to be readily repairable via postnatal skeletal remodeling, are not mechanistically linked to malformation, and often are seen in the presence of maternal or fetal toxicity. As such, these minor variations would not generally be considered adverse in and of themselves but should be interpreted in the context of other maternal and fetal findings, information available on normal skeletogenesis patterns, mode of action of the test agent, and historical control incidence using a weight of evidence approach.     

Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P pI incorporation into at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP3), by selectively binding PIP2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP3-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation.     

Identification of Pseudomonas alcaligenes chromosomal DNA in the plasmid DNA of the chlorobenzene-degrading recombinant Pseudomonas putida strain CB1-9.

The recombinant Pseudomonas putida strain CB1-9, which acquired the ability to grow on chlorobenzenes, contains a 33-kilobase (kb) plasmid (pKFL3) which lacked homology to an indigenous 15-kb plasmid (pKFL1) in Pseudomonas alcaligenes C-0 parent but was homologous to a 55-kb plasmid (pKFL2) from the P. putida R5-3 parent. Chromosomal DNA of P. alcaligenes C-0 hybridized to probes prepared from pKFL3 but not to probes prepared from pKFL2. A single clone from a genomic library of P. alcaligenes C-0 hybridized to EcoRI-digested pKFL3. Southern blot hybridization with the insert DNA from that clone identified homology with specific restriction enzyme fragments in pKFL3. The ability of the recombinant to utilize 3-chlorobenzoate, chlorobenzene, and 1,4-dichlorobenzene as well as its loss of utilization of xylenes and methylbenzoates appears to be associated with the transfer and integration of chromosomal DNA from P. alcaligenes into a Tol-like plasmid of P. putida R5-3.     

Electron microscopy evidence that cytoplasmic localization of the p16INK4A "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

CPDNA INHERITANCE IN INTERSPECIFIC CROSSES AND EVOLUTIONARY INFERENCE IN LOUISIANA IRISES

Inheritance of chloroplast DNA haplotypes was determined for progeny from interspecific crosses involving Iris fulva and Iris hexagona. Polymerase chain reaction amplification of chloroplast DNA followed by restriction fragment length analysis of the amplification products was used to identify the haplotypes of 213 experimental hybrids. This analysis allowed a test for maternal, paternal, and biparental inheritance in the hybrid offspring. Two of the hybrid progeny possessed haplotypes that were combinations of those present in the pollen and seed parents. One of the offspring possessed only the paternal haplotype. The remaining 210 plants had the haplotypes characteristic of the maternal plant. Chloroplast DNA variation in iris populations has previously been used to infer not only introgressive hybridization between I. fulva and I. hexagona, but also the greater role of direct pollen transfer relative to seed dispersal as the avenue for interspecific gene flow. We reexamined the previous conclusions concerning the mode of introgressive hybridization between I. fulva and I. hexagona in light of the results from the chloroplast DNA inheritance analysis. The low level of paternal and biparental inheritance detected in this analysis suggests that previous analyses using the chloroplast DNA as a seed-specific marker were robust. Furthermore, data concerning barriers to hybridization between I. fulva and I. hexagona suggest that the probability of chloroplast DNA introgression via pollen is low.     

Nitrogen and phosphorus dynamics and retention in ecotones of Lake Titicaca,Bolivia/Peru

We are studying present conditions and consequences of material movement from land to water in the Lake Titicaca basin, and how fluxes are affected by human activities. The principal objective of this research is to describe and explain the variability in the Andean Altiplano of (a) water, nutrient and sediment fluxes from land and (b) composition, nutrient limitation and other important features of nearshore lake communities, and compare the effects of different agricultural practices (especially traditional and modern) on these factors. We are focusing on a comparison of the impacts of two forms of agriculture in this region: ancient raised fields currently under rehabilitation, and flat pastures and fields, which are more common. Results of the first year of study indicate there is substantial variability in nitrogen and phosphorus dynamics in relation to ecotone complexity (simple vs. intermediate vs. complex). Raised field sites have the beneficial effect of reducing high available nutrient concentrations (nitrate and soluble reactive phosphorus) and sediment load (measured as turbidity) as the water passes through them enroute to the lake. Aquatic vegetation (algae and macrophytes) reflect well ambient total nitrogen and phosphorus concentrations. Experimental nutrient limitation bioassays indicate that nitrogen is the most important limiting nutrient, though there is important spatial variability within the landscape, and phosphorus as well as nitrogen can be limiting.