Functional human transcobalamin II isoproteins are secreted by insect cells using the baculovirus expression system

Transcobalamin II (TCII) is a cobalamin (Cbl, vitamin B12)-binding protein in mammalian plasma that facilitates the cellular uptake of the vitamin. To obtain human TCII in sufficient quantity for analytical studies, the complementary DNA (cDNA) encoding TCII was inserted into the plasmid PVL 1393, and the baculovirus expressing TCII was obtained by homologous recombination in Spodoptera frugiperda (SF9) insect cells by cotransfection with the wildtype virus. Under optimized conditions, SF9 cells infected with the recombinant virus secreted 2 to 4 micrograms of TCII per milliliter of culture medium. TCII did not accumulate in the SF9 cells and seemed to be constitutively secreted as observed previously in cultured human endothelial cells. The purified recombinant TCII has the same molecular weight by SDS-PAGE as purified human TCII. The recombinant TCII cross-reacts with an antiserum to native human TCII, binds Cbl and facilitates the uptake of Cbl in eukaryotic cells by binding to the receptor for TCII-Cbl on the plasma membrane of K562 cells. Amino acid sequence analysis of the purified recombinant TCII identified two polypeptides, one identical to the amino acid sequence deduced from the cDNA and a second lacking the first and second N-terminal residues. These sequences are identical to two TCII polypeptides purified from Cohn fraction III of pooled human plasma. The two forms of recombinant TCII have the same isoelectric points as the two predominant isoprotein forms of TCII in human serum. Since the baculovirus construct contains a single cDNA that can encode only one amino acid sequence, the two isoproteins in recombinant TCII must be generated by a mechanism other than allele specific expression. A plausible mechanism for generating isoproteins of nonglycosylated peptides, such as TCII, may be by splicing of the leader peptide at alternative sites.     

Lopinavir protein binding in HIV‐1‐infected pregnant women

Background

Pregnancy may alter protein binding (PB) of highly bound protease inhibitors due to changes in plasma concentrations of albumin and α‐1 acid glycoprotein (AAG). Small changes in PB can greatly impact the fraction of drug unbound (FU) exerting pharmacological effect. We report lopinavir (LPV) PB during third trimester (antepartum, AP) compared to ≥1.7 weeks postpartum (PP) to determine if FU changes compensate for reduced total concentrations reported previously.

Methods

P1026s enrolled women receiving LPV/ritonavir, soft gel capsules 400/100 mg or 533/133 mg twice daily. LPV FU, albumin and AAG were determined AP and PP.

Results

AP/PP samples were available from 29/25 women respectively with all but one woman receiving the same dose AP/PP. LPV FU was increased 18% AP vs. PP (mean 0.96±0.16% AP vs. 0.82±0.21% PP, P=0.001). Mean protein concentrations were reduced AP (AAG=477 mg/L; albumin=3.28 mg/dL) vs. PP (AAG=1007 mg/L; albumin=3.85 mg/dL) (P<0.0001 for each comparison). AAG concentration correlated with LPV binding. Total LPV concentration did not correlate with LPV FU AP or PP. However, higher LPV concentration PP was associated with reduced PB and higher FU after adjustment for AAG.

Conclusions

LPV FU was higher and AAG lower AP vs. PP. The 18% increase in LPV FU AP is smaller than the reduction in total LPV concentration reported previously and is not of sufficient magnitude to eliminate the need for an increased dose during pregnancy.

     

DJ‐1 is a Parkinson's disease susceptibility gene in southern Italy

De Marco EV, Annesi G, Tarantino P, Nicoletti G, Civitelli D, Messina D, Annesi F, Arabia G, Salsone M, Condino F, Novellino F, Provenzano G, Rocca FE, Colica C, Morelli M, Scornaienchi V, Greco V, Giofrè L, Quattrone A. DJ‐1 is a Parkinson's disease susceptibility gene in southern Italy. Mutations in the gene DJ‐1 have been shown to be a rare cause of early‐onset Parkinson's disease (EOPD). Since DJ‐1 mutations have been found in patients with Parkinson's disease (PD) from southern Italy, we aimed to investigate whether polymorphisms within the DJ‐1 gene could represent a risk factor for sporadic PD. First, we genotyped 294 patients with PD and 298 controls coming from southern Italy to assess the distribution of the insertion/deletion (Ins/Del) polymorphism. In a second phase, we identified five single‐nucleotide polymorphisms (SNPs) useful to delimit a region potentially involved and genotyped all patients and controls for these markers. All the markers analyzed were significantly associated with PD at both allelic and genotypic level. The most significant association with the disease was found at the Ins/Del polymorphism (p = 0.0001; adjusted odds ratio (OR ) = 2.05; confidence interval (CI ) = 1.36–3.08). When we considered a three‐marker sliding window, we found a highly significant association between the disease and the haplotypes including markers rs17523802, Ins/Del, and rs3766606 (p = 0.0007) and markers Ins/Del, rs3766606 and rs7517357 (p = 0.0054). Our results indicate that polymorphisms located in a region spanning 3535 bp from the promoter to the intron 2 of the DJ‐1 gene confer risk to sporadic PD in southern Italy.     

Genotype of the cystic fibrosis population of the Hunter Region of New South Wales

Objective : To determine the genotype of patients attending the cystic fibrosis clinic at John Hunter Hospital, Newcastle, Australia.
Methodology : Seventy-five of the 76 patients attending the clinic over a 6 month period had blood collected for genetic analysis of 17 of the cystic fibrosis (CF) gene mutations.
Results : Sixty-one per cent of the patients were homozygous for the ΔF508 mutation and all except one child had at least one ΔF508 mutation.
Discussion : Nearly 80% of the CF genes were the ΔF508 mutation. This prevalence suggests that the obligatory false negative rate of a newborn screening programme for CF based on a combination of immunoreactive trypsin and the ΔF508 gene may be as low as 4-5%.     

CSF rhinorrhea: detection and localization using overpressure cisternography with Tc-99m-DTPA

We performed 32 overpressure radionuclide cisternography (ORNC) studies to examine 26 patients who were clinically suspected of having cerebrospinal fluid (CSF) fistula with rhinorrhea. Fifteen (47%) of these cisternography studies were positive, and the site of the leak was identified. No leak could be demonstrated in the other 17. Of 23 examinations performed in patients who had clinically documented CSF rhinorrhea, 15 (65%) were scintigraphically positive. The rapid cephalad transit of the radionuclide bolus allowed completion of the study within 30 to 45 minutes. Seven examinations were also performed with overpressure metrizamide CT cisternography (OMCTC), and five demonstrated concordant results with the radionuclide study. Patient discomfort and side effects were minimal. We conclude that radionuclide infusion cisternography is a safe, rapid, and accurate method of investigating a suspected or proven CSF rhinorrhea and that it is complementary to metrizamide cisternography.