Lipovitellin constitutes the protein backbone of glycoproteins involved in sperm-egg interaction in the amphibian Discoglossus pictus.

Our knowledge of the molecules that interact with sperm at the egg membrane is restricted to a short list. In the eggs of Discoglossus pictus, fusion with sperm is limited to a differentiated structure, the dimple, offering several advantages for detecting molecules involved in fertilization. Previous studies have identified fucosylated glycoproteins of 200, 260, and 270 kDa located at the surface of the dimple that are able to bind sperm in vitro. Here, we show that dimple glycoproteins and a protein represented by a 120-kDa band released following gel-into-gel SDS-PAGE of both glycoproteins share the same N-terminal amino acid sequence, which itself is similar to the N-termini of Xenopus liver-synthesized vitellogenin (VTG) and the lipovitellin 1. MALDI/MS mass spectrometry indicated that the 120-kDa band is part of both gps 200 and 270/260. A 117-kDa major protein of the egg lysate exhibits the same MALDI/MS spectrum, and LC-MSMS indicates that this is a lipovitellin 1 (DpLIV) that coincides with the 120-kDa band and is responsible for the formation of the 200-270-kDa dimers. Therefore, lipovitellin 1 constitutes the protein backbone of the dimple glycoconjugates. In vitro assays using polystyrene beads coated with DpLIV or with its dimers indicate that significant sperm binding occurs only with DpLIV dimers. In amphibians, VTG is taken up by the oocyte, where it releases lipovitellins destined to form yolk. In Discoglossus, our data suggest that yolk proteins are also synthesized by the oocyte. The dimple forms in the ovulated oocyte following the exocytosis of vesicles that likely expose DpLIVs at their membrane. Indeed, in whole mounts of immunostained eggs, anti-vitellogenin antibodies label only the surface of the dimple.

Blood p50 evaluation enhances diagnostic definition of isolated erythrocytosis.

BACKGROUND: High oxygen-affinity haemoglobin variants and 2,3-diphosphoglycerate (2,3-DPG) deficiency are inherited diseases generating low tissue oxygen tension and erythropoietin-driven erythrocytosis, that characterizes the clinical phenotype of patients. Level of blood p50 (the oxygen tension at which haemoglobin is 50% saturated) is used to recognize these conditions. OBJECTIVES: To define the clinical utility of blood p50 measurement in the diagnosis of isolated erythrocytosis. SUBJECTS AND DESIGN: Venous blood p50 measurement was included in the diagnostic work-up of 102 consecutive patients with isolated erythrocytosis besides blood cell count, arterial oxygen saturation, serum erythropoietin measurement and screening for JAK2 mutations. SETTING: Haematological Outpatient Section at University Hospital. RESULTS: Seven patients had relative erythrocytosis. Among 95 patients with absolute erythrocytosis, 4 (4.2%) had decreased p50 level. The extended study of family members revealed a familial inheritance. Two families had haemoglobin variants already described as Haemoglobin Malmö and Haemoglobin San Diego. In one family, the proband had a new high oxygen-affinity haemoglobin variant (Haemoglobin Safi) resulting from the transversion C-->A at codon 81 of the alpha2-globin gene. In the last family, a deficiency of 2,3-DPG was found. Within the 91 patients with normal p50 values, 46 (51%) had secondary erythrocytosis, 13 (14%) polycythemia vera and 32 (35%) idiopathic erythrocytosis. CONCLUSIONS: This study suggests that the investigation of blood p50 level may be useful to define diagnosis in patients with isolated erythrocytosis.

Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor.

Nerve growth factor (NGF) is a member of the neurotrophin family. These growth factors support neuronal survival and differentiation. Neurotrophins are synthesized as pre-pro-proteins. Whereas the pre-sequences mediate secretion, the function of the pro-peptides is largely unknown. To test the role of the pro-sequence as a folding enhancer, recombinant human pro-NGF (rh-pro-NGF) was produced in Escherichia coli. The oxidative refolding of rh-pro-NGF and rh-NGF was studied using electrospray mass spectrometry (ESIMS) time-course analysis. This analysis permitted both the identification and quantification of intermediates present during the process. The disulfide bonds formed at different times of the refolding processes were characterized by proteolytic digestion followed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) analysis. Folding yields and kinetics of rh-pro-NGF were significantly enhanced when compared to the in vitro refolding of mature rh-NGF. These results suggest that the pro-sequence of NGF promotes folding of the mature part.

Effects of pyridoxine-pyrrolidon-carboxylate on hepatic and cerebral ATP levels in ethanol treated rats.

ATP levels were measured in the liver and brain of rats acutely intoxicated with ethanol. The pretreatment of the animals with pyridoxine-pyrrolidon carboxylate (Metadoxine) prevented the marked fall in ATP concentration caused by ethanol in both organs.

[Quantitative evaluation of HbA2. Comparison of 3 methods]. / Valutazione quantitativa della HbA2. Comparazione di tre metodiche.

Two electrophoretic tests and one based on column chromatographic method have been studied for quantitative and qualitative evaluation of HbA2. Results of 1069 assays demonstrated a satisfactory qualitative resolution for the three methods but for the best quantitative precision, the test of choice is the chromatographic one.