Characterization and Genetic Study of the Ovine α S2 -Casein (CSN1S2) Allele B

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine α _s2 -casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp₇₅ → Tyr₇₅ and Ile₁₀₅ → Val₁₀₅. The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp₇₅ → Tyr₇₅ substitution modifies the protein electric charge, milk properties may result affected to some extent.     

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA(+) /DAPI (-) heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA (+) regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.     

Dilutional control of prothrombin activation at physiologically relevant shear rates

The generation of proteolyzed prothrombin species by preassembled prothrombinase in phospholipid-coated glass capillaries was studied at physiologic shear rates (100–1000 s⁻¹). The concentration of active thrombin species (α-thrombin and meizothrombin) reaches a steady state, which varies inversely with shear rate. When corrected for shear rate, steady-state levels of active thrombin species exhibit no variation and a Michaelis-Menten analysis reveals that chemistry of this reaction is invariant between open and closed systems; collectively, these data imply that variations with shear rate arise from dilutional effects. Significantly, the major products observed include nonreactive species arising from the loss of prothrombin's phospholipid binding domain (des F1 species). A numerical model developed to investigate the spatial and temporal distribution of active thrombin species within the capillary reasonably approximates the observed output of total thrombin species at different shears; it also predicts concentrations of active thrombin species in the wall region sufficient to account for observed levels of des FI species. The predominant feedback formation of nonreactive species and high levels of the primarily anticoagulant intermediate meizothrombin (∼40% of total active thrombin species) may provide a mechanism to prevent thrombus propagation downstream of a site of thrombosis or hemorrhage.     

Molecular recognition between Azotobacter vinelandii rhodanese and a sulfur acceptor protein

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.     

Hydroxyapatite affinity chromatography for the highly selective enrichment of mono‐ and multi‐phosphorylated peptides in phosphoproteome analysis

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi‐phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono‐, di‐, tri‐, and multi‐phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.     

Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.     

Telomerase repeat amplification protocol (TRAP): A new molecular marker for parathyroid carcinoma.

Telomerase results to be active in human germ, stem cells, several malignant cell tumors and in immortalized cell lines. In order to investigate if molecular mechanisms other than Rb gene inactivation can be helpful to diagnose malignancy of parathyroid tumors, we decided to investigate the presence of active telomerase in homogenates from different pathological parathyroid tissues (hyperplastic, adenomatous, carcinomatous, and normal) and primary cell cultures. The TRAP assay was performed to detect this activity in histologically characterized normal, hyperplastic, adenomatous, and carcinomatous human parathyroid tissues, primary cell lines, and one metastatic tissue from parathyroid carcinoma. Only malignant parathyroid glands and the metastatic tissue were TRAP positive. Our findings suggest that telomerase expression could represent an important molecular mechanism underlying the acquisition and progression of an aggressive phenotype of epithelial parathyroid cells and it may help to predict their malignant potential. The TRAP assay is easy to perform and it could become an additional tool to be included in the harmamentarium for the molecular diagnosis of parathyroid carcinoma.