Structural characterization of the L0 cytoplasmic loop of human multidrug resistance protein 6 (MRP6)

ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E₂₀₅-G₂₇₉ contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. ¹H NMR spectroscopy identified three helical regions separated by more flexible regions.     

Shedding of the 67-kD laminin receptor by human cancer cells

The 67-kD laminin receptor (67LR) is a cell membrane-associated molecule exhibiting high affinity for the basement membrane glycoprotein, laminin. While export of the 67LR toward the extracellular matrix has been recently suggested by electron microscopy studies, there is to date no evidence of shedding of the 67LR from cells. Using two monoclonal antibodies directed against the 67LR, we developed a double-determinant radioimmunoassay that demonstrates that the 67LR is released from cancer cells into the culture medium. The shed molecule exhibited the same apparent molecular weight as that of the membrane-associated 67LR, suggesting that no proteolytic cleavage is involved in the process. Furthermore, we demonstrate that the 67LR is not anchored to the membrane through a glycolsyl-phosphatidylinositol bridge. However, the observation that lactose increased the release of 67LR suggests that a lectin-type interaction is involved in the cell membrane association of this laminin binding protein and the cell surface. Interestingly, the released 67LR recovered after HPLC gel filtration was found free as well as associated to high molecular weight complexes. The free 67LR retained its ability to bind to the cell surface. Our study is the first demonstration that the 67LR is effectively shed by cancer cells. The released free 67LR could play an important role in modulating interactions between cancer cells and laminin during tumor invasion and metastasis. © 1996 Wiley-Liss, Inc.     

Synthesis and enantioselectivity of the enantiomers of PG9 and SM21, new potent analgesic and cognition-enhancing drugs

The enantiomers of two α-tropanyl esters, SM₂₁ (1) and PG₉ (2), derived from (+)-R-hyoscyamine, that act by increasing the central cholinergic tone, were obtained by esterification after resolution of the corresponding racemic acids [(−)-S-1, (−)-R-2 and (+)-S-2] and by stereospecific synthesis [(+)-R-1]. Their analgesic and cognition-enhancing activities were tested in mice and their ACh-releasing properties determined on rat parietal cortex. These compounds show enantioselectivity in analgesic and cognition-enhancing tests on mice, the eutomers being the isomers which possess the same spatial arrangement of the groups on the chiral atom as (+)-R hyoscyamine [(+)-R-SM₂₁, (+)-S-PG₉]. The ACh-releasing effect of the enantiomers of SM₂₁ in rats is in agreement with the results in mice, while PG₉ enantiomers do not show any appreciable enantioselectivity in this test. On the basis of the different effects of the 5-HT₄ antagonist SDZ 205557 on analgesia induced by the enantiomers of 1 and 2 and by (+)-R-hyoscyamine and the α-tropanyl ester of 2-phenylpropionic acid 3, a mechanism of action is proposed for this class of compounds. © 1996 Wiley-Liss, Inc.     

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L⁻¹ of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment. Received: 12 June 1997 / Accepted: 24 October 1997     

Involvement of tyrosine kinase activity in 1alpha,25(OH)2-vitamin D3 signal transduction in skeletal muscle cells

In cultured chick skeletal muscle cells loaded with Fura-2, the tyrosine kinase inhibitors herbimycin A and genistein abolished both the fast inositol 1,4,5-trisphosphatedependent Ca(2+) release from internal stores and extracellular Ca(2+) influx induced by 1alpha, 25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)). Daidzein, an inactive analog of genistein, was without effects. Tyrosine phosphatase inhibition by orthovanadate increased cytosolic Ca(2+). Anti-phosphotyrosine immunoblot analysis revealed that 1alpha, 25(OH)(2)D(3) rapidly (0.5-10 min) stimulates in a concentrationdependent fashion (0.1-10 nm) tyrosine phosphorylation of several myoblast proteins, among which the major targets of the hormone could be immunochemically identified as phospholipase Cgamma (127 kDa), which mediates intracellular store Ca(2+) mobilization and external Ca(2+) influx, and the growth-related proteins mitogen-activated protein (MAP) kinase (42/44 kDa) and c-myc (65 kDa). Genistein suppressed the increase in phosphorylation and concomitant elevation of MAPK activity elicited by the sterol. Both genistein and the MAPK kinase (MEK) inhibitor PD98059 abolished stimulation of DNA synthesis by 1alpha,25(OH)(2)D(3). The sterol-induced increase in tyrosine phosphorylation of c-myc, a finding not reported before for cell growth regulators, was totally suppressed by the specific Src inhibitor PP1. These results demonstrate that tyrosine phosphorylation is a previously unrecognized mechanism involved in 1alpha,25(OH)(2)D(3) regulation of Ca(2+) homeostasis in hormone target cells. In addition, the data involve tyrosine kinase cascades in the mitogenic effects of 1alpha, 25(OH)(2)D(3) on skeletal muscle cells.     

Efficacy of transfer factor in treating patients with recurrent ocular herpes infections

Recurrent ocular herpes is an insoluble problem for the clinician. As cellular immunity plays an important role in controlling herpes relapses, and other studies have shown the efficacy of HSV-specific transfer factor (TF) for the treatment of herpes patients, an open clinical trial was undertaken in 134 patients (71 keratitis, 29 kerato-uveitis, 34 uveitis) suffering from recurrent ocular herpetic infections. The mean duration of the treatment was 358 days, and the entire follow-up period 189121 before, and 64062 days after TF treatment. The cell-mediated immune response to the viral antigens, evaluated by the lymphocyte stimulation test (LST) and the leucocyte migration test (LMT) (P<0.001), was significantly increased by the TF treatment. The total number of relapses was decreased significantly during/after TF treatment, dropping from 832 before, to 89 after treatment, whereas the cumulative relapse index (RI) dropped, during the same period, from 13.2 to 4.17 (P<0.0001). No side effects were observed. It is concluded that patients with relapsing ocular herpes can benefit from treatment with HSV-specific TF.     

Classification of European Mtdnas from an Analysis of Three European Populations

Mitochondrial DNA (mtDNA) sequence variation was examined in Finns, Swedes and Tuscans by PCR amplification and restriction analysis. About 99% of the mtDNAs were subsumed within 10 mtDNA haplogroups (H, I, J, K, M, T, U, V, W, and X) suggesting that the identified haplogroups could encompass virtually all European mtDNAs. Because both hypervariable segments of the mtDNA control region were previously sequenced in the Tuscan samples, the mtDNA haplogroups and control region sequences could be compared. Using a combination of haplogroup-specific restriction site changes and control region nucleotide substitutions, the distribution of the haplogroups was surveyed through the published restriction site polymorphism and control region sequence data of Caucasoids. This supported the conclusion that most haplogroups observed in Europe are Caucasoid-specific, and that at least some of them occur at varying frequencies in different Caucasoid populations. The classification of almost all European mtDNA variation in a number of well defined haplogroups could provide additional insights about the origin and relationships of Caucasoid populations and the process of human colonization of Europe, and is valuable for the definition of the role played by mtDNA backgrounds in the expression of pathological mtDNA mutations     

Sex Differences in Territorial Behavior Exhibited by the Spotted Hyena (Hyaenidae, Crocuta crocuta)

Spotted hyenas ( Crocuta crocuta ) are gregarious carnivores that defend group territories against encroachment by neighboring conspecifics. Here we monitored the behavior of members of one clan of free-ranging spotted hyenas during border patrols, 'wars' with neighboring clans, and other interactions with alien intruders, to document differences between the sexes in territorial behavior in this species. We also examined the possibility that the probability or rate of attack on alien hyenas encountered within the clan's territory would vary with the sex of the intruders. Initiation and leadership of most cooperative territorial behaviors were by adult female clan members, although border patrols were occasionally conducted by groups composed exclusively of resident males. The vast majority of alien intruders into the territory of the study clan were males. Resident females were more likely to attack intruding females than intruding males, but hourly rates of aggression directed by females towards aliens did not vary with intruder sex. Resident males were more likely than resident females to attack alien males, and resident males directed significantly higher hourly rates of aggression towards intruding males than females. Although female leadership in most cooperative territorial behaviors distinguishes spotted hyenas from many mammalian carnivores, other sex differences in the territorial behavior of spotted hyenas resemble those documented in other gregarious predators. Sex differences observed in hyena territoriality are consistent with the hypothesis that male and female clan members derive different selective benefits from advertisement and defense of group territories.     

Pyrrolizidine alkaloid profiles of the Senecio cineraria group (Asteraceae)

Alkaloid profiles of five Senecio species (Asteraceae), including S. ambiguus subsp. ambiguus, S. ambiguus subsp. nebrodensis, S. gibbosus subsp. bicolor, S. gibbosus subsp. gibbosus, and S. gibbosus subsp. cineraria, were studied. Eleven pyrrolizidine alkaloids were identified and their content was evaluated by GLC-MS and GLC analysis. Otosenine and florosenine were found to be the major alkaloids in all studied species. It is interesting that only S. ambiguus subsp. nebrodensis was characterized by a high content of the alkaloids jacobine, jacoline, jaconine, and jacozine.