Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Allelic Variation,Aneuploidy, and Nongenetic Mechanisms Suppress a Monogenic Trait in Yeast

Clinically relevant features of monogenic diseases, including severity of symptoms and age of onset, can vary widely in response to environmental differences as well as to the presence of genetic modifiers affecting the trait’s penetrance and expressivity. While a better understanding of modifier loci could lead to treatments for Mendelian diseases, the rarity of individuals harboring both a disease-causing allele and a modifying genotype hinders their study in human populations. We examined the genetic architecture of monogenic trait modifiers using a well-characterized yeast model of the human Mendelian disease classic galactosemia. Yeast strains with loss-of-function mutations in the yeast ortholog (GAL7) of the human disease gene (GALT) fail to grow in the presence of even small amounts of galactose due to accumulation of the same toxic intermediates that poison human cells. To isolate and individually genotype large numbers of the very rare (∼0.1%) galactose-tolerant recombinant progeny from a cross between two gal7Δ parents, we developed a new method, called “FACS-QTL.” FACS-QTL improves upon the currently used approaches of bulk segregant analysis and extreme QTL mapping by requiring less genome engineering and strain manipulation as well as maintaining individual genotype information. Our results identified multiple distinct solutions by which the monogenic trait could be suppressed, including genetic and nongenetic mechanisms as well as frequent aneuploidy. Taken together, our results imply that the modifiers of monogenic traits are likely to be genetically complex and heterogeneous.     

Effects of aerobic exercise therapy and cognitive behavioural therapy on functioning and quality of life in amyotrophic lateral sclerosis: protocol of the FACTS-2-ALS trial

Background

Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain.

Methods

We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles.

Results

Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct.

Conclusions

The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.     

Genetic variation in wholesale carcass cuts predicted from digital images in cattle

The objective of this study was to quantify the genetic variation in carcass cuts predicted using digital image analysis in commercial cross-bred cattle. The data set comprised 38,404 steers and 14,318 heifers from commercial Irish herds. The traits investigated included the weights of lower value cuts (LVC), medium value cuts (MVC), high value cuts (HVC), very high value cuts (VHVC) and total meat weight. In addition, the weights of total fat and total bones were available on the steers. Heritability of carcass cut weights, within gender, was estimated using an animal linear model, whereas genetic and phenotypic correlations among cuts were estimated using a sire linear model. Carcass weight was included as a covariate in all models. In the steers, heritability ranged from 0.13 (s.e. = 0.02) for VHVC to 0.49 (s.e. = 0.03) for total bone weight, and in the heifers heritability ranged from 0.15 (s.e. = 0.04) for MVC to 0.72 (s.e. = 0.06) for total meat weight. The coefficient of genetic variation for the different cuts varied from 1.4% to 3.6%. Genetic correlations between the different cut weights were all positive and ranged from 0.45 (s.e. = 0.08) to 0.89 (s.e. = 0.03) in the steers, and from 0.47 (s.e. = 0.14) to 0.82 (s.e. = 0.06) in the heifers. Genetic correlations between the wholesale cut weights and carcass conformation ranged from 0.32 (s.e. = 0.06) to 0.45 (s.e. = 0.07) in the steers, and from 0.10 (s.e. = 0.12) to 0.38 (s.e. = 0.09) in the heifers. Genetic correlations between the same wholesale cut traits in steers and heifers ranged from 0.54 (s.e. = 0.14) for MVC to 0.79 (s.e. = 0.06) for total meat weight; genetic correlations between carcass weight and carcass classification for conformation and fat score in both genders varied from 0.80 to 0.87. The existence of genetic variation in carcass cut traits, coupled with the routine availability of predicted cut weights from digital image analysis, clearly shows the potential to genetically improve carcass value.     

Activation of an alternative, rec12 (spo11)-independent pathway of fission yeast meiotic recombination in the absence of a DNA flap endonuclease

Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Delta mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Delta rec12Delta strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms.