The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

Physical microhabitat requirements of freshwater pearl mussels,Margaritifera margaritifera (L.)

Surface sediment diatoms from the east coast of Lake Tanganyika were analysed using ordination and classification techniques, and compared with assemblages previously described from the northern part of the lake. Grain-size analyses were performed on subsamples. Four groups of diatom assemblages were recognised. The first group clusters samples taken in the north, far from the Rusizi river mouth. The second group comprises samples taken on silty sediment along the Tanzanian coast, including one sample taken near the mouth of the Malagarazi river and those from the northernmost part of the lake. The third group comprises surface sediments along the Burundian coast (near Ramba and Magara), and the fourth is characterised by epipsammic taxa. A sample taken near the central arm of the Malagarazi river is included in the latter group. The impact of small rivers on the diatom assemblages in the surface sediments is restricted to the mouth area.     

Products of mitochondrial protein synthesis in Tetrahymena.

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57,500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform-methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.     

Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.     

Further aspects of IL-1 beta secretion revealed by transfected monkey kidney cells.

Because the cytokine interleukin-1 beta (IL-1 beta) lacks a classical hydrophobic signal sequence, it has been unclear how it is released from cells, and whether release proceeds via a novel mechanism or through non-specific leakage. To address this issue, we have examined the secretion of the recombinant forms of human IL-1 beta from COS monkey kidney cells, which express low levels of endogenous IL-1 beta. Four proteins were expressed: precursor and mature IL-1 beta and precursor and mature IL-1 beta fused to an amino terminal hydrophobic signal sequence from human tissue plasminogen activator. By monitoring the appearance of a known cytosolic protein (ATP citrate lyase) in the medium, we find that the unmodified IL-1 beta s are non-specifically released in very small quantities from the cytosol. On the other hand, the signal sequence-modified IL-1 beta s are glycosylated and efficiently secreted by the ER/Golgi pathway. The secreted, modified-mature protein is also biologically active, suggesting that this pathway has been bypassed for reasons other than maintaining the structural integrity of IL-1 beta. More likely the alternative pathway is a critical aspect of IL-1 biology. The differences in kinetics and quantity of IL-1 beta release from monocytic and COS cells suggest that COS cells lack critical components for the rapid release seen in monocytes.