Subcellular targeting and function of osteoblast nucleotide pyrophosphatase phosphodiesterase 1

The ectonucleoside pyrophosphatase phosphodiesterase 1 (NPP1/PC-1) is a member of the NPP enzyme family that is critical in regulating mineralization. In certain mineralizing sites of bone and cartilage, membrane-limited vesicles [matrix vesicles (MVs)] provide a sheltered internal environment for nucleation of calcium-containing crystals, including hydroxyapatite. MV formation occurs by budding of vesicles from the plasma membrane of mineralizing cells. The MVs are enriched in proteins that promote mineralization. Paradoxically, NPP1, the type II transmembrane protein that generates the potent hydroxyapatite crystal growth inhibitor inorganic pyrophosphate (PP_i), is also enriched in MVs. Although osteoblasts express NPP1, NPP2, and NPP3, only NPP1 is enriched in MVs. Therefore, this study uses mineralizing human osteoblastic SaOS-2 cells, a panel of NPP1 mutants, and NPP1 chimeras with NPP3, which does not concentrate in MVs, to investigate how NPP1 preferentially targets to MVs. We demonstrated that a cytosolic dileucine motif (amino acids 49–50) was critical in localizing NPP1 to regions of the plasma membrane that budded off into MVs. Moreover, transposition of the NPP1 cytoplasmic dileucine motif and flanking region (AAASLLAP) to NPP3 conferred to NPP3 the ability to target to the plasma membrane and, subsequently, concentrate in MVs. Functionally, the cytosolic tail dileucine motif NPP1 mutants lost the ability to support MV PP_i concentrations and to suppress calcification. The results identify a specific targeting motif in the NPP1 cytosolic tail that delivers PP_i-generating NPP activity to osteoblast MVs for control of calcification. calcification; dileucine motif; NPP3     

Repellency of essential oils of some Kenyan plants against Anopheles gambiae

Essential oils of six plants growing in Kenya were screened for repellent activities against Anopheles gambiae sensu stricto. The oils of Conyza newii (Compositeae) and Plectranthus marrubioides (Labiateae) were the most repellent (RD50=8.9 x 10(-5) mg cm(-2), 95% CI) followed by Lippia javanica (Verbenaceae), Lippia ukambensis (Verbenaceae), Tetradenia riparia, (Iboza multiflora) (Labiateae) and Tarchonanthus camphoratus (Compositeae). Eight constituents of the different oils (perillyl alcohol, cis-verbenol, cis-carveol, geraniol, citronellal, perillaldehyde, caryophyllene oxide and a sesquiterpene alcohol) exhibited relatively high repellency. Four synthetic blends of the major components (present in > or = 1.5%) of the essential oils were found to exhibit comparable repellent activity to the parent oils.     

Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.     

Excitation energy transport and trapping in concentrated solid solutions of flavomononucleotide

Excitation energy transport and trapping is studied for monomer-fluorescent dimer system of flavomononucleotide (FMN) in polyvinyl alcohol films (PVA). It is shown that the theory neglecting reverse energy transfer (RET) from dimers to monomers does not allow for the explanation of concentration quenching and concentration depolarization results presented herein. Much better agreement has been obtained using generalized energy transport theory in which fluorescent dimers are treated as imperfect traps for excitation energy. Such parameters like the dimer quantum yield and its emission anisotropy are estimated.     

Xenobiotic conjugation systems in deer compared with cattle and rat

The ability of cattle and deer liver to catalyse xenobiotic conjugation reactions was investigated and compared with that of the rat. Marked differences in the activity of these enzymes were noted between the domestic animals and rats. Hepatic microsomal epoxide hydrolase activity in cattle and deer, determined using benzo[a]pyrene 4,5-oxide as substrate, was nearly twice that of the rat. In contrast, glutathione S-transferase activity in hepatic cytosol, determined with 1-chloro-2,4-dinitrobenzene as substrate, was significantly lower in the cattle and deer. When 1,2-dichloro-4-nitrobenzene served as the accepting substrate, no activity was detectable in the cattle and deer. Similarly, glutathione reductase activity and total glutathione levels were markedly lower in the cattle and deer compared with the rat. Cytosolic sulfotransferase activity, monitored using 2-naphthol as substrate, was higher in cattle compared with the rat. Finally, microsomal UDP-glucuronosyl transferase activity, determined using 1-napththol as substrate, did not differ significantly among the three species.     

Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system

The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.     

Is p53 intronic variant G13964C associated with predisposition to cancer?

Germline mutations of the p53 gene confer a high risk of diverse malignancies. The highest frequency of inherited p53 defects was noted in Li-Fraumeni syndrome (LFS), but almost half of the mutations were found in families with incomplete Li-Fraumeni-like syndrome (LFL), including familial breast cancer cases. Recently, a germline intronic G13964C base change of the p53 was reported as a high-risk mutation associated with familial breast cancer (LEHMAN et al. 2000). We genotyped Polish cancer patients and healthy control individuals for the G13964C variant. Patients were chosen from cancer families with phenotypes typical for germline mutations of p53 (LFS, LFL), BRCA1 [hereditary breast (ovarian) cancer, HB(O)C] or a complex consistent with both LFL and HB(O)C. Children with leukemia were included in the study as another high risk group (FELIX et al. 1992). The G13964C variant was detected in six of 87 (6.9%) cancer patients (including two ALL children), but also in eight of 96 (8.3%) control individuals (p > 0.4). Thus we found no evidence of the variant's association with a high risk of cancer.