ADAM 10: an active metalloprotease expressed during avian epithelial morphogenesis

The ADAMs are a family of proteins containing multiple functional domains. We have cloned the avian orthologue of ADAM 10 and demonstrate that it has metalloprotease activity. Chick ADAM 10 is expressed in the developing dermatome and myotome of the somite, epidermis, gut endoderm, the epithelial tissues of the kidney, liver, and heart, and in neural crest cells. The expression patterns and protein distribution of ADAM 10 suggest it may play a significant role in the morphogenesis of several epithelial tissues. When a dominant-negative metalloprotease-mutant form of ADAM 10 is expressed in the ectoderm or ADAM 10 expression is knocked down with morpholinos, morphogenesis and tissue specification are altered.     

In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function

We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.     

Common variations in noncoding regions of the human natriuretic peptide receptor A gene have quantitative effects

Genetic susceptibility to common conditions, such as essential hypertension and cardiac hypertrophy, is probably determined by various combinations of small quantitative changes in the expression of many genes. NPR1, coding for natriuretic peptide receptor A (NPRA), is a potential candidate, because NPRA mediates natriuretic, diuretic, and vasorelaxing actions of the nariuretic peptides, and because genetically determined quantitative changes in the expression of this gene affect blood pressure and heart weight in a dose-dependent manner in mice. To determine whether there are common quantitative variants in human NPR1, we have sequenced the entire human NPR1 gene and identified 10 polymorphic sites in its non-coding sequence by using DNA from 34 unrelated human individuals. Five of the sites are single nucleotide polymorphisms; the remaining five are length polymorphisms, including a highly variable complex dinucleotide repeat in intron 19. There are three common haplotypes 5' to this dinucleotide repeat and three 3' to it, but the 5' haplotypes and 3' haplotypes appear to be randomly associated. Transient expression analysis in cultured cells of reporter plasmids with the proximal promoter sequences of NPR1 and its 3' untranslated regions showed that these polymorphisms have functional effects. We conclude that common NPR1 alleles can alter expression of the gene as much as two-fold and could therefore significantly affect genetic risks for essential hypertension and cardiac hypertrophy in humans.     

Neuropeptides modulate rat chorda tympani responses

The neuropeptide leptin has been shown to selectively modulate rat chorda tympani (CT) responses to sweet tastants. To explore whether other neuropeptides can modulate such responses, rat whole nerve CT responses to NaCl, HCl, quinine HCl, and sucrose were measured while administering cholecystokinin-8 (CCK-8), substance P(4-11) (SP(4-11)), or calcitonin gene-related peptide (CGRP). To avoid possible confounding effects on CT responses that take long times to develop, such as those that arise from intraperitoneal injections, we investigated the effects of the above peptides injected into the ipsilateral lingual artery (LA) on CT nerve responses during the initial seconds after a tastant was placed on the tongue. We found that CT responses to NaCl and HCl were increased by CCK-8 and decreased by CGRP. SP(4-11) had no noticeable effect. Peptide-induced CT responses to quinine HCl or sucrose were too small to accurately detect. These data suggest that at short latencies, after local infusion via the LA, neuropeptides can alter CT responses in a peptide-specific manner.     

Modeling jet fuel (JP-8) fate and transport in soils with plants

Vegetation is often used to clean up soils and groundwater contaminated with organic contaminants. Plant-induced upward water movement may draw organic contaminants spilled near the watertable to the more aerated near-surface soil. The objective of this study was to develop and verify a 1-D model of fate and transport of JP-8, a kerosene-based jet fuel, in soil. The modeling approach considered the advective and dispersive transport of jet fuels dissolved in groundwater, which may undergo simple first-order decay or linear adsorption. The governing partial differential advection dispersion equation was solved in one dimension. Data from an experiment of fate and transport of JP-8 with plant-induced upward water movement were used to verify the model. Simulated results with different scenarios described the experimental results well for different depths above the contaminated zone in both vegetated and unvegetated columns. Advection was the dominant mechanism near the contaminated zone and advection with retardation and decay was used to fit the data away from the contaminated zone. Results indicated that the soil water movement impacted the transport and concentration of JP-8 in the soil columns. This model can be used to simulate the fate of JP-8 associated with phytoremediation and evapotranspiration.     

Technical note: fate and transport of jet fuel (JP-8) in soils with selected plants

Remediation of soil and groundwater contaminated by leaking fuel storage tanks may be assisted by plants, although plant effects on abiotic and biotic removal processes remain unclear. The objectives of this study were to investigate abiotic and biotic removal of JP-8, a kerosene-based jet fuel, in soils with plants, and to determine the effects of plant-induced water movement. Loss of JP-8 in a dry-soil, control column was 25% after 5 months, primarily due to volatilization and gas-phase diffusion. By comparison, managed treatments with simulated surface spills averaged 86% mass reduction at 5 months, indicating an important contribution of biodegradation. Overall JP-8 mass reduction was similar in surface and subsurface-irrigated systems, indicating water content, not mode of water application, influences bioremediation in near-surface systems. The JP-8 concentration reductions in soil columns contaminated above a simulated watertable were 36% after 3 months and 50% after 12 months for vegetated columns compared to 26% and 34% in unplanted columns. Downward movement of JP-8 in unplanted columns was double that in planted columns. Near the groundwater table, JP-8 persists longer than near the soil surface. Plants promote upward movement of water and help draw spilled JP-8 to aerobic near-surface soil.     

Localization of a class III myosin to filopodia tips in transfected HeLa cells requires an actin-binding site in its tail domain

Bass Myo3A, a class III myosin, was expressed in HeLa cells as a GFP fusion in order to study its cellular localization. GFP-Myo3A localized to the cytoplasm and to the tips of F-actin bundles in filopodia, a localization that is consistent with the observed concentration toward the distal ends of F-actin bundles in photoreceptor cells. A mutation in the motor active site resulted in a loss of filopodia localization, suggesting that Myo3A motor activity is required for filopodial tip localization. Deletion analyses showed that the NH2-terminal kinase domain is not required but the CO2H-terminal 22 amino acids of the Myo3A tail are required for filopodial localization. Expression of this tail fragment alone produced fluorescence associated with F-actin throughout the cytoplasm and filopodia and a recombinant tail fragment bound to F-actin in vitro. An actin-binding motif was identified within this tail fragment, and a mutation within this motif abolished both filopodia localization by Myo3A and F-actin binding by the tail fragment alone. Calmodulin localized to filopodial tips when coexpressed with Myo3A but not in the absence of Myo3A, an observation consistent with the previous proposal that class III myosins bind calmodulin and thereby localize it in certain cell types.     

Synthesis and preliminary biological evaluation of 6-O-[11C]-[(methoxymethyl)benzyl]guanines,new potential PET breast cancer imaging agents for the DNA repair protein AGT

Novel radiolabeled O(6)-benzylguanine derivatives, 6-O-[(11)C]-[(methoxymethyl)benzyl]guanines ([(11)C]p-O(6)-MMBG, 1a; [(11)C]m-O(6)-MMBG, 1b; ([(11)C]o-O(6)-MMBG, 1c), have been synthesized for evaluation as new potential positron emission tomography (PET) breast cancer imaging agents for DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT).     

Colloidal Calcium Phosphate in the Reconstituted Milk Micelle May Direct Wild-type Recombinant Human β-Casein to Fold Like the Native Protein

Native human β-casein (CN) at all phosphorylation levels exhibits reproducible behavior and appears to have a unique, stable folding pattern. In contrast, the recombinant non-phosphorylated form of human β-CN (β-CN-0P) with the exact amino acid sequence (wild-type), expressed and purified from Escherichia coli, differs greatly in its behavior from the native protein and the complexes formed are unstable to thermal cycling. However, when it was incorporated into reconstituted milk micelles, using bovine κ-CN at a κ/β molar ratio of 1/3 with added Ca²⁺ ions and inorganic phosphate (P_i) at levels that would ordinarily precipitate, its association behavior vs. temperature as monitored by turbidity (OD_{400 nm}) approximated that of native β-CN-0P. This suggests that the milk micelle system, and particularly the colloidal calcium phosphate, may act as a ‘molecular chaperon’ to direct the folding of the molecule into the highly stable conformation found in the purified native human β-CN molecule.