EARLY DIAGNOSIS OF MALIGNANT MELANOMA OF THE SKIN

About five per cent of all malignant lesions of the skin are malignant melanomas. The poor prognosis associated with this malignant lesion emphasizes the importance of early diagnosis. A large proportion of malignant melanomas arise in preexisting lesions such as junction nevi, precancerous melanoses and, much more rarely, blue nevi. Early malignant changes in these precursor lesions include increasing pigmentation, enlargement, thickening, crusting, bleeding, ulceration, tumor formation, and development of satellite lesions.Many pigmented, and some non-pigmented, lesions of the skin must be differentiated from malignant melanoma. Since even with radical surgical treatment the prognosis of malignant melanoma is poor, junction nevi which are subject to continual trauma or have signs of probable malignant degeneration should be prophylactically excised.     

Isolation and genetic characterization of the porcine apolipoprotein E gene

The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (ape-E) gene. A single positive recombinant phage clone containing a 10·7-kb insert was isolated from a porcine genomic library, and a 4·2-kb fragment was subcloned and sequenced. The 4·2-kb fragment contained the entire apo-E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo-E gene is made up of four exons and three introns, and encodes a preapo-E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo-E gene contains a (CG)₁₃ microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo-E gene to chromosome 6 band q2·1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.     

Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.     

Use of embryonic and somatic cells for production of transgenic domestic animals

In contrast to the highly developed genetic modification systems available for manipulating the mouse genome, at this time only simple gain of function modifications can be undertaken in domestic species. Clearly, the greatest barrier to gene targeting in domestic species has been the unavailability of cell lines that can be modified in vitro and still be used to generate a living organism. In the mouse, the embryonic stem (ES) cells and embryonic germ (EG) cells have fulfilled that role. While the nuclear transfer procedures have solved this problem in sheep and cattle, in swine ES and EG cells are still needed. In addition, targeting in domestic species is affected by the need to develop targeting constructs containing isogenic DNA regions. As a result, it is necessary to isolate the gene of interest, sequence required regions, and develop isogenic targeting constructs by technologies such as long-range PCR. On the positive side, enrichment protocols developed in the mouse can be applied to domestic species, thus facilitating the identification of correctly modified cell lines. Hence, progress in mammalian cloning, the development of EG cell lines, and advances in gene targeting presently allows the introduction of precise genetic modifications into the domestic animal genome.     

The genetic architecture of complex traits in teosinte (Zea mays ssp. parviglumis): new evidence from association mapping

Previous association analyses showed that variation at major regulatory genes contributes to standing variation for complex traits in Balsas teosinte, the progenitor of maize. This study expands our previous association mapping effort in teosinte by testing 123 markers in 52 candidate genes for association with 31 traits in a population of 817 individuals. Thirty-three significant associations for markers from 15 candidate genes and 10 traits survive correction for multiple testing. Our analyses suggest several new putative causative relationships between specific genes and trait variation in teosinte. For example, two ramosa genes (ra1 and ra2) associate with ear structure, and the MADS-box gene, zagl1, associates with ear shattering. Since zagl1 was previously shown to be a target of selection during maize domestication, we suggest that this gene was under selection for its effect on the loss of ear shattering, a key domestication trait. All observed effects were relatively small in terms of the percentage of phenotypic variation explained (<10%). We also detected several epistatic interactions between markers in the same gene that associate with the same trait. Candidate-gene-based association mapping appears to be a promising method for investigating the inheritance of complex traits in teosinte.     

Virus-like particles as quantitative probes of membrane protein interactions

We demonstrate that virus-like particles carrying conformationally complex membrane proteins ("lipoparticles") can be used as soluble probes of membrane protein interactions. To demonstrate the utility of this approach, we use lipoparticles to rapidly differentiate the relative kinetics of membrane protein interactions using optical biosensor technology. The technique is applied to diverse membrane proteins, including G protein-coupled receptors, and used to rank the relative kinetics of nearly all the commercially available monoclonal antibodies against chemokine receptor CCR5. These particles serve as versatile probes for screening crude and purified antibody preparations for receptor specificity, epitope reactivity, and relative binding kinetics.     

Fine mapping of a quantitative resistance gene for gray leaf spot of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) derived from teosinte (<Emphasis Type="Italic">Z. mays</Emphasis> ssp. <Emphasis Type="Italic">parviglumis</Emphasis>)

Key message

In this study we mapped the QTL Qgls8 for gray leaf spot (GLS) resistance in maize to a ~130 kb region on chromosome 8 including five predicted genes.

Abstract

In previous work, using near isogenic line (NIL) populations in which segments of the teosinte (Zea mays ssp. parviglumis) genome had been introgressed into the background of the maize line B73, we had identified a QTL on chromosome 8, here called Qgls8, for gray leaf spot (GLS) resistance. We identified alternate teosinte alleles at this QTL, one conferring increased GLS resistance and one increased susceptibility relative to the B73 allele. Using segregating populations derived from NIL parents carrying these contrasting alleles, we were able to delimit the QTL region to a ~130 kb (based on the B73 genome) which encompassed five predicted genes.

     

Vitamin D insufficiency in a population of healthy western Canadians

BackgroundPeople with low levels of vitamin D and its metabolites are at increased risk for osteoporotic fractures. We wished to ascertain levels of vitamin D in a representative sample of adult western Canadians, to help assess the level of risk. We evaluated the prevalence of vitamin D insufficiency, defined as 25-hydroxyvitamin D [25(OH)D] less than 40 nmol/L, and seasonal variations in 25(OH)D, parathyroid hormone and related biochemical indices in a community-dwelling population of healthy Canadians living in Calgary (lati- tude 51°07''N).MethodsDuring calendar year 1999, we collected fasting overnight blood samples every 3 months from 60 men and 128 women (age range 27 to 89 years) who had volunteered to participate in another study. We used commercial radioimmunoassay kits to measure calciotrophic hormones and other biochemical indices. Regression models for longitudinal data were used to assess the effect of season and other potential predictors on individual parameters.ResultsFor a total of 64 participants (34%), vitamin D insufficiency, defined as 25(OH)D less than 40 nmol/L, was recorded at least once out of the 4 sampling times. After adjustment for age, body mass index and holiday travel, we observed the anticipated rise in serum 25(OH)D from a mean of 57.3 (standard deviation [SD] 21.3) nmol/L in the winter to 62.9 (SD 28.8) nmol/L in spring (p = 0.001) and 71.6 (SD 23.6) nmol/L in summer (p < 0.001), with a subsequent decline to 52.9 (SD 17.2) nmol/L in the fall (p = 0.008). The anticipated inverse relation between 25(OH)D and parathyroid hormone was not consistently observed: after adjustment for age, sex, body mass index and serum calcium, serum levels of parathyroid hormone did decrease significantly, from 39.5 (SD 18.8) ng/L in winter to 36.3 (SD 17.8) ng/L in summer (p = 0.001), but they continued to decline to 34.5 (SD 17.3) ng/L in the fall (p < 0.001). There was no association between 25(OH)D and parathyroid hormone (p = 0.21).InterpretationWe documented a high prevalence of vitamin D insufficiency, which warrants consideration of dietary vitamin D supplementation.Vitamin D deficiency has long been recognized as a cause of rickets in children and osteomalacia in adults. More recent is the awareness of a preclinical phase of vitamin D deficiency, known as vitamin D insufficiency, which increases the risk of osteoporotic fractures.^1,2,3 Low levels of vitamin D metabolites are associated with malabsorption of calcium, which results in bone loss.^4,5 Vitamin D can be obtained through the diet or it is synthesized in the skin after exposure to the sun. However, because few foods provide a natural source of vitamin D⁶ and because fortification of foods with vitamin D is often unreliable,^7,8 skin synthesis is thought to constitute the major source. People living in countries at higher latitudes are more prone to seasonal vitamin D insufficiency because wintertime sunlight does not promote conversion of the vitamin D precursor in the skin.⁹ Levels of vitamin D and its main circulating metabolite, 25-hydroxy vitamin D [25(OH)D], are under the predominant influence of solar ultraviolet B radiation (wavelength 290 to 315 nm). Webb and colleagues⁹ have shown that in Boston (latitude 42°N), sun irradiation is inadequate to generate previtamin D in vitro from November through February; in Edmonton (latitude 53°3$\acute{0}$N), this period extends from October through March.The vitamin D hormone system and parathyroid hormone are the principal regulators of serum concentrations of calcium and therefore influence skeletal calcium reserves. Because of the health risks associated with low levels of vitamin D, our objective was to determine patterns of seasonal variation in 25(OH)D, as well as the prevalence of vitamin D insufficiency, defined as 25(OH)D less than 40 nmol/L,¹⁰ in a population of healthy men and women living in western Canada. Although 1,25-dihydroxy vitamin D [1,25-(OH)₂D] is the most potent form of vitamin D, 25(OH)D is the main circulating metabolite of vitamin D and is considered the correct functional indicator of vitamin D stores in humans.^11,12 We also examined associations between 25(OH)D and serum concentrations of 1,25-(OH)₂D, parathyroid hormone and other related biochemical markers.     

Host-guest scale of left-handed polyproline II helix formation

Despite the clear importance of the left-handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix-forming propensities measured for all residues, except tyrosine and tryptophan, in a proline-based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix-forming propensity scale will prove useful in future studies of the conformational properties of proline-rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states.