Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal -tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the -tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.     

An investigation of the storage and biosynthesis of phenylpropenes in sweet basil

Plants that contain high concentrations of the defense compounds of the phenylpropene class (eugenol, chavicol, and their derivatives) have been recognized since antiquity as important spices for human consumption (e.g. cloves) and have high economic value. Our understanding of the biosynthetic pathway that produces these compounds in the plant, however, has remained incomplete. Several lines of basil (Ocimum basilicum) produce volatile oils that contain essentially only one or two specific phenylpropene compounds. Like other members of the Lamiaceae, basil leaves possess on their surface two types of glandular trichomes, termed peltate and capitate glands. We demonstrate here that the volatile oil constituents eugenol and methylchavicol accumulate, respectively, in the peltate glands of basil lines SW (which produces essentially only eugenol) and EMX-1 (which produces essentially only methylchavicol). Assays for putative enzymes in the biosynthetic pathway leading to these phenylpropenes localized many of the corresponding enzyme activities almost exclusively to the peltate glands in leaves actively producing volatile oil. An analysis of an expressed sequence tag database from leaf peltate glands revealed that known genes for the phenylpropanoid pathway are expressed at very high levels in these structures, accounting for 13% of the total expressed sequence tags. An additional 14% of cDNAs encoded enzymes for the biosynthesis of S-adenosyl-methionine, an important substrate in the synthesis of many phenylpropenes. Thus, the peltate glands of basil appear to be highly specialized structures for the synthesis and storage of phenylpropenes, and serve as an excellent model system to study phenylpropene biosynthesis.     

Nitric oxide inhibits execution of apoptosis at two distinct ATP-dependent steps upstream and downstream of mitochondrial cytochrome c release

The endogenous mediator nitric oxide (NO) blocked apoptosis of Jurkat cells elicited by staurosporine, anti-CD95 or chemotherapeutics, and switched death to necrosis. The switch in the mode of cell death was dependent on the ATP loss elicited by NO. This affected two distinct steps of the apoptotic cascade. First, the release of cytochrome c from mitochondria was delayed by NO. Second, processing of procaspases-3/7 to the active proteases was prevented even after cytochrome c had been released. Thus, NO interferes with execution steps of apoptosis both upstream and downstream of cytochrome c release.     

Elemental composition of human milk from mothers of premature and full-term infants during the first 3 months of lactation

To examine longitudinal and gestational effects of mineral content in human milk, we analyzed human milk from lactating mothers of premature (PRT,n = 24, < 2000g birth weight, < 37 wk gestation) and fullterm (FT,n = 19, > 2500g, 39–41 wk gestation), living in Newfoundland, Canada. Samples were collected once a week for 8 wk with one final sample collected at 3 mo. Milk samples collected in acid-washed containers were wet ashed with concentrated HNO₃, and barium, cadmium, calcium, cesium, cobalt, copper, cerium, lanthanum, magnesium, manganese, molybdenum, nickel, lead, rubidium, tin, strontium, and zinc were measured using inductively coupled plasma-mass spectrometry. Data were analyzed using standard multiple-regression procedures with correlated data analyses to take account of the relationship between successive weeks. Results indicated lower Ca and Pb in PRT milk. Calcium was the only nutritionally significant element to differ between groups. Molybdenum in both PRT and FT milk showed a definite decrease with time, suggesting that the Mo content in milk is homeostatically regulated. However, Ce, La, Ba, and Sn did not display any pattern indicative of biological regulation and potential human requirement.     

Eosinophil peroxidase increases membrane permeability in mammalian urinary bladder epithelium

Eosinophilperoxidase (EPO), a cationic protein found in eosinophils, has beenreported to be cytotoxic independent of its peroxidase activity. Thisstudy investigated with electrophysiological methods whether EPO istoxic to mammalian urinary bladder epithelium. Results indicate thatEPO, when added to the mucosal solution, increases apical membraneconductance of urinary bladder epithelium only when the apical membranepotential is cell interior negative. The EPO-induced conductance wasconcentration dependent, with a maximumconductance of 411 µS/cm² and aMichaelis-Menten constant of 113 nM. The EPO-induced conductance wasnonselective for K⁺ andCl. The conductance waspartially reversed using voltage but not by removal of EPO from thebulk solution. Mucosal Ca²⁺reversed the EPO-induced conductance by a mechanism involving reversible block of the conductance. Prolonged exposure (up to 1 h) toEPO was toxic to the urinary bladder epithelium, as indicated by anirreversible increase in transepithelial conductance. These resultssuggest that EPO is indeed toxic to urinary bladder epithelium via amechanism that involves an increase in membrane permeability.     

A phage display selection of engrailed homeodomain mutants and the importance of residue Q50

Mutants of engrailed homeodomain (HD) that retain DNA-binding activity were isolated using a phage display selection. This selection was used to enrich for active DNA-binding clones from a complex library consisting of over a billion members. A more focused library of mutant homeodomains consisting of all possible amino acid combinations at two DNA-contacting residues (I47 and Q50) was constructed and screened for members capable of binding tightly and specifically to the engrailed consensus sequence, TAATTA. The isolated mutants largely recapitulated the distribution of amino acids found at these positions in natural homeodomains thus validating the in vitro selection conditions. In particular, the unequivocal advantage enjoyed by glutamine at residue 50 is surprising in light of reports that minimize the importance of this residue. Here, the subtle contributions of residue Q50 are demonstrated to play a functionally important role in specific recognition of DNA. These results highlight the complex subtlety of protein–DNA interactions, underscoring the value of the first reported in vitro selection of a homeodomain.