Myocardial Inflammatory Changes Before and After Antiretroviral Therapy Initiation in People With Advanced Human Immunodeficiency Virus Disease.

Because of the high frequency of late presentation of human immunodeficiency virus (HIV) disease in our population, we decided to explore the presence of myocarditis among people with HIV infection and advanced immunosuppression (less than 200 CD4+ cells/µL) and to describe the inflammatory changes observed after combined antiretroviral therapy initiation in an observational, longitudinal, prospective cohort. We performed both cardiovascular magnetic resonance imaging and doppler transthoracic echocardiogram.

Effect of marine by-product meals on hen egg production parameters, yolk lipid composition and sensory quality.

The effect of including 5% marine by-product meals in feeds of laying hens on egg production, composition and sensory characteristics was tested. Marine by-product meals were prepared using two methods: (i) cooking (100°C/10 min) followed by drying (60°C/24 hr) or (ii) grinding followed by drying. The raw materials used for meal production were scallop or squid viscera, shrimp heads or whole mackerel. A total of 108 laying hens were allocated to nine diet treatments; one control diet (corn and soya bean based) and eight experimental diets, containing 95% of the control feed and 5% of the experimental meal for three weeks. Daily intake was higher in hens fed the dried mackerel and cooked shrimp meals. All the experimental treatments showed significantly higher concentration of n-3 HUFA in yolk reserves and phospholipids compared to the control (0.12-0.13 g per 100 g), especially those with scallop or squid prepared by both methods (0.53-0.95 g per 100 g). Scallop, squid and shrimp meal inclusion in the feed produced eggs with more astaxanthin (0.22 mg per 100 g) while this carotenoid was absent in the control and mackerel treatments. Visual evaluation of raw yolk colour increased with the inclusion of marine by-product meals with higher values in hens fed shrimp heads (13), followed by scallop viscera (11), squid viscera (9), and with similar values for mackerel and control (4). The taste, aroma, texture and colour of cooked eggs from different treatments were not statically different when evaluated by a panel of 60 untrained people. These results suggest that meals from marine by-products are a better alternative for improving egg yolk composition by increasing n-3 HUFA when compared to fishmeal as they also increase astaxanthin and yolk pigmentation without affecting egg sensory characteristics.

Proteolytic cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa.

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antichicken alpha/beta-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth.

Analysis of structure and microtubule assembly activity of the Drosophila 205K MAP.

Phosphorylation, dimerization and binding to calmodulin have been reported to influence the microtubule assembly capacities of MAPs (microtubule-associated proteins). Here we report that the Drosophila 205K MAP is a phosphoprotein in vivo and can be phosphorylated by cdc2/p34 in vitro. Bacterially produced 205K MAP is competent of microtubule assembly and microtubule bundling and binds to immobilized calmodulin in a Ca2+-dependent way. EM rotary shadowing analyses suggest that 205K MAP consists of an amino-terminal flexible extended region and a carboxy-terminal globular domain. This carboxy-terminal region harbors the microtubule binding site and sequences required for dimerization, as confirmed by in vitro crosslinking experiments of truncated proteins.

Molecular cloning and expression studies of two divergent alpha-tubulin genes in Neurospora crassa.

Three alpha-tubulin isoforms were previously detected in Neurospora crassa. We have cloned and analysed two alpha-tubulin cDNAs, Tub alpha A and Tub alpha B that encode polypeptides of 453 and 451 amino acids, respectively. The encoded amino acids exhibit an unusual divergence of 35%. This is the highest divergence ever observed between alpha-tubulins from the same species. The expression of the two genes is developmentally regulated. We did not detect any transcription of the Tub alpha A gene in dormant macroconidia and during the first 30 min of development even though the alpha-tub A protein is already present in the early stage of germination. In contrast, the Tub alpha B gene is continuously transcribed during the vegetative cycle and the expression profile of the protein follows the ones of its mRNA.

Developmental changes in the(45)Ca (2+) uptake byTrichoderma viride mycelium.

The rate of the(45)Ca(2+) uptake by the submergedTrichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the(45)Ca(2+) uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of(45)Ca(2+) uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up(45)Ca(2+) proportionally with time. The autoradiography of colonies showed that(45)Ca(2+) was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of(45)Ca(2+) in conidia.

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa.

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.

Identification and partial purification of calmodulin-binding microtubule-associated proteins from Neurospora crassa.

We have purified microtubule-associated proteins from Neurospora crassa on the basis of heat stability and affinity to calmodulin. Two proteins of molecular masses 170 kDa and 190 kDa have been partially purified. A third protein of 145 kDa was purified almost to homogeneity, and we present evidence that this protein is a specific substrate for a Ca2+/calmodulin-dependent protein kinase. The purified 170-, 190-, and 145-kDa proteins induce the assembly of microtubules from purified porcine brain tubulin. We demonstrate that all three proteins are microtubule-associated proteins on the basis of an in vitro microtubule-binding assay.

Molecular cloning of a cDNA encoding calmodulin from Neurospora crassa.

A full-length cDNA encoding Neurospora crassa calmodulin was isolated from a lambda ZAP II cDNA expression library. The open reading frame encodes a protein of 148 amino acid residues with a calculated M(r) of 16,865 Da. Using site-directed mutagenesis, the complete cDNA was ligated into a trc promoter-regulated bacterial expression vector to allow expression of N. crassa calmodulin in E. coli. The expressed protein was found to be identical to the native protein on the basis of some of its biochemical properties. Finally, Southern analysis of restriction digests of genomic DNA indicates that calmodulin is encoded by a single-copy gene.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurospora crassa.

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.