Rapid blood separation is superior to fluoride for preventing in vitro reductions in measured blood glucose concentration.

AIMS: To determine whether tubes containing sodium fluoride negatively bias blood glucose concentration by directly comparing glucose concentrations in paired blood samples collected in tubes containing lithium heparin (Li-Heparin) and tubes containing sodium fluoride/potassium oxalate (NaF-KOx). METHODS: Paired blood samples from a group of patients (n = 1040) were collected in tubes containing Li-Heparin and tubes containing NaF-KOx at the same time. All Li-Heparin samples were centrifuged soon after collection and were kept cool in transport along with NaF-KOx samples, which were centrifuged at the receiving location after an average transport time of 4 h, but immediately before analysis. Glucose concentrations in the paired samples were determined simultaneously by an automated oxidase method. RESULTS: The mean glucose concentrations for NaF-KOx samples and Li-Heparin samples were 5.7 mmol/l and 6.1 mmol/l, respectively, with a mean difference of 0.39 mmol/l. CONCLUSION: Rapid separation of heparinised blood is superior to fluoride alone for abrogating glycolytic effects on blood glucose measurements in the clinical laboratory.

Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles.

Intraflagellar transport (IFT) is a motility in which particles composed of at least 17 polypeptides move underneath the flagellar membrane. Anterograde (outward) and retrograde (inward) movements of these IFT particles are mediated by FLA10 kinesin-II and cytoplasmic dynein DHC1b, respectively. Mutations affecting IFT particle polypeptides or motors result in the inability to assemble flagella. IFT particles and the motors moving them are located principally around the basal bodies as well as in the flagella. Here, we clone the cDNA encoding one of the IFT particle proteins, IFT52, and show by immunofluorescence that while some IFT52 is in the flagella, the majority is found in two horseshoe-shaped rings around the basal bodies. Immunoelectron microscopy indicates that IFT52 is associated with the periphery of the transitional fibers, which extend from the distal portion of the basal body to the cell membrane and demarcate the entrance to the flagellar compartment. This localization suggests that the transitional fibers form a docking complex for the IFT particles destined for the flagellum. Finally, the flagellaless mutant bld1 completely lacks IFT52 due to a deletion in the gene encoding IFT52.

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella.

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.

Mutations in specific I-A(k) alpha(2) and beta(2) domain residues affect surface expression.

A previous investigation demonstrated that several mutations in class II dimer-of-dimers contact residues interfere with antigen presentation by transfectants but not with plasma membrane expression of the mutant class II. In the present study we examined other class II mutations in this region that did inhibit plasma membrane expression of mutant class II molecules. Molecules containing both mutations H alpha 181D in the alpha(2) domain and E beta 170K in the beta(2) domain exhibited low plasma membrane expression, but molecules with only one of these mutations were expressed normally. The mutant class II molecules were transported to organelles that were accessible to a fluid-phase protein, hen egg lysozyme (HEL). Culture of transfectants with lysozyme enhanced the amount of class II compact dimer (alpha beta plus peptide; CD), and this was especially marked for the class II mutant H alpha 181D/E beta 170K and for other molecules possessing both mutations. Formation of class II CD was not paralleled by an increase in class II surface expression. Thus the joint mutation of H alpha 181 and E beta 170 has two effects. In the absence o high concentrations of exogenous peptide, it prevents efficient CD formation, possibly by affecting invariant chain (Ii) proteolysis and/or the stability of the class II after Ii/CLIP is removed. At high peptide concentrations supplied by exogenous HEL, the mutations allow CD formation, but not expression of class II on the plasma membrane. Molecular modeling of the possible interaction of class II and Ii suggests that the mutant amino acids H alpha 181D and E beta 170K, besides affecting the overall stability of class II, might also interact with Ii via two loops in class II's alpha(2) and beta(2) domains respectively.