Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.     

Carbamoyl-phosphate synthetases from Neurospora crassa. Immunological relatedness of the enzymes from Neurospora, bacteria, yeast, and mammals

Neurospora crassa contains two carbamoyl-phosphate synthetases: a mitochondrial enzyme (CPS-A) which supplies carbamoyl phosphate for arginine biosynthesis, and a nuclear enzyme whose product is used for the synthesis of pyrimidines. We have prepared antiserum against a highly purified preparation of the large subunit of CPS-A and have used the antiserum to demonstrate that the large subunit is, like most mitochondrially localized proteins, initially synthesized as a higher molecular weight precursor. The CPS-A antiserum cross-reacts with the nuclear enzyme, allowing us to identify the product of the complex N. crassa pyr-3 genetic locus as a protein with a subunit molecular weight of 180,000. Finally, we have found that the CPS-A antiserum also cross-reacts with carbamoyl-phosphate synthetases from bacteria, yeast, and mammals. The immunological relatedness of carbamoyl-phosphate synthetases from such diverse species suggests that the protein sequences required for carbamoyl phosphate production have been highly conserved during the course of evolution.     

A catalogue of <Emphasis Type="Italic">Triticum monococcum</Emphasis> genes encoding toxic and immunogenic peptides for celiac disease patients

The celiac disease (CD) is an inflammatory condition characterized by injury to the lining of the small-intestine on exposure to the gluten of wheat, barley and rye. The involvement of gluten in the CD syndrome has been studied in detail in bread wheat, where a set of “toxic” and “immunogenic” peptides has been defined. For wheat diploid species, information on CD epitopes is poor. In the present paper, we have adopted a genomic approach in order to understand the potential CD danger represented by storage proteins in diploid wheat and sequenced a sufficiently large number of cDNA clones related to storage protein genes of Triticum monococcum. Four bona fide toxic peptides and 13 immunogenic peptides were found. All the classes of storage proteins were shown to contain harmful sequences. The major conclusion is that einkorn has the full potential to induce the CD syndrome, as already evident for polyploid wheats. In addition, a complete overview of the storage protein gene arsenal in T. monococcum is provided, including a full-length HMW x-type sequence and two partial HMW y-type sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.     

Impact of the TCR Signal on Regulatory T Cell Homeostasis,Function, and Trafficking

Signaling through the T cell antigen receptor (TCR) is important for the homeostasis of naïve and memory CD4⁺ T cells. The significance of TCR signaling in regulatory T (Treg) cells has not been systematically addressed. Using an Ox40-cre allele that is prominently expressed in Treg cells, and a conditional null allele of the gene encoding p56^Lck, we have examined the importance of TCR signaling in Treg cells. Inactivation of p56^Lck resulted in abnormal Treg homeostasis characterized by impaired turnover, preferential redistribution to the lymph nodes, loss of suppressive function, and striking changes in gene expression. Abnormal Treg cell homeostasis and function did not reflect the involvement of p56^Lck in CD4 function because these effects were not observed when CD4 expression was inactivated by Ox40-cre.The results make clear multiple aspects of Treg cell homeostasis and phenotype that are dependent on a sustained capacity to signal through the TCR.     

Modeling the effects of El Niño, density-dependence, and disturbance on harbor seal (Phoca vitulina) counts in Drakes Estero, California: 1997–2007

Harbor seal ( Phoca vitulina ) haul-out site use may be affected by natural or anthropogenic factors. Here, we use an 11-yr (1997–2007) study of a seal colony located near a mariculture operation in Drakes Estero, California, to test for natural (El Niño-Southern Oscillation (ENSO), density-dependence, long-term trends) and anthropogenic (disturbance or displacement related to oyster production activities) factors that may influence the use of haul-out subsites. Annual mariculture related seal disturbance rates increased significantly with increases in oyster harvest ( r _s= 0.55). Using generalized linear models (GLMs) ranked by best fit and Akaike's Information Criteria, ENSO and oyster production (as a proxy for disturbance/displacement) best explained the patterns of seal use at all three subsites near the mariculture operations, with effects being stronger at the two subsites closest to operations. Conversely, density-dependence and linear trend effects poorly explained the counts at these subsites. We conclude that a combination of ENSO and mariculture activities best explain the patterns of seal haul-out use during the breeding/pupping season at the seal haul-out sites closest to oyster activities.     

Germination,reproduction and interference in the amphicarpic annual Emex spinosa (L.) Campd

Summary A comparison was made of the dimorphic subterranean and aerial achenes of the annual Emex spinosa (L.) Campd. Subterranean achenes were less dormant, had a higher percentage of viability, germinated at a faster rate, were less temperature-dependent in germination and produced larger seedlings than aerial achenes. Subterranean achenes germinated, equally well in light or dark but aerial achenes had a strong light requirement. There was a linear relationship between numbers of weight of aerial achenes and plant size in the field. More resources were allocated to reproduction in plants restricted in size because of environmental conditions. Subterranean achene production was less affected than aerial by density, nitrogen levels, varying field conditions and interference between plants grown, from subterranean and aerial achenes. In mixtures of such plants, those from subterranean achenes had larger leaf area and stems and more aerial achenes. These differences were not apparent in monocultures by the time of harvest. Seed dimorphism is more likely in short-lived fugitive species and the different roles associated with dimorphism (persistence in situ versus dispersal in E. spinosa) are seen as an ecological advantage in such species.