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Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583-20590; Nomizu et al. [1996] FEBS Lett. 396:37-42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730 and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin alpha2 chain-specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide-treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly. 相似文献
85.
The type I restriction-modification system EcoR124I recognizes and binds to the split DNA recognition sequence 5'-GAAN(6)RTCG-3'. The methyltransferase, consisting of HsdM and HsdS subunits with the composition M2S, can interact with one or more subunits of the HsdR subunit to form the endonuclease. The interaction of the methyltransferase with HsdR has been investigated by surface plasmon resonance, showing that there are two non-equivalent binding sites for HsdR which differ in binding affinity by at least two orders of magnitude. DNA footprinting experiments using Exonuclease III suggest that the addition of HsdR to the methyltransferase (at a stoichiometry of either 1:1 or 2:1) increases the stability of the resulting DNA-protein complex but does not increase the size of the footprint. More extensive in situ footprinting experiments using copper-phenanthroline on the DNA-protein complexes formed by M2S, R1M2S and R2M2S also show no difference in the detailed cleavage pattern, with approximately 18 nucleotides protected on both strands in each complex. Thus the HsdR subunit(s) of the endonuclease stabilise the interaction of the M2S complex with DNA, but do not directly contribute to DNA binding. In addition, the thymidine nucleotide in the tetranucleotide recognition sequence GTCG is hyper-reactive to cleavage in each case, suggesting that the DNA structure in this region is altered in these complexes. 相似文献
86.
VR Lord JW Cherwonogrodzky GG Schurig RD Lord MJ Marcano GE Meléndez 《Canadian Metallurgical Quarterly》1998,59(5):546-551
We have investigated the inter-relationship between the opioid and aminergic systems in the control of secretion of the pro-oestrous LH surge and the involvement of delta-opioid receptor subtypes in this process. Conscious female rats bearing a cannula in the femoral artery were injected i.p. with a selective delta-opioid receptor agonist (DPDPE) either alone or with the opioid antagonist (naloxone) at 1300 h on the day of pro-oestrus. Blood samples were collected hourly between 1500 h and 1900 h, and plasma LH levels were measured by RIA. At the end of this period (1900 h), the animals were autopsied and the concentrations of the amines (noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT)) and their metabolites (dihydroxyphenolglycol (DHPG) and 5-hydroxyindoleacetic acid (5HIAA), metabolites of NA and 5HT respectively) were determined by HPLC with electrochemical detection in the medial preoptic area, suprachiasmatic nucleus, median eminence and arcuate nucleus. DPDPE abolished the LH surge and concomitantly decreased hypothalamic NA and DHPG concentrations in all the areas examined. The levels of DA, 5HT and 5HIAA were also reduced in all hypothalamic regions studied, except DA and 5HIAA in the suprachiasmatic nucleus. Naloxone reversed these inhibitory effects of the delta-agonist. We conclude that activation of delta-opioid receptors may exert an inhibitory effect on LH release. The effect is probably an indirect one mediated by the monoaminergic systems, as they are suppressed by DPDPE in nearly all the hypothalamic regions studied. 相似文献
87.
SL Anliker MS McClure TC Britton EA Stephan SR Maple GG Cooke 《Canadian Metallurgical Quarterly》1994,83(5):716-719
The anti-tumor agent gemcitabine hydrochloride, a beta-difluoronucleoside, is remarkably stable in the solid state. In 0.1 N HCI solution at 40 degrees C, deamination of gemcitabine occurs, yielding its uridine analogue. Approximately 86% of the initial gemcitabine remains after 4 weeks under these conditions. Cleavage of the N-glycosidic bond of gemcitabine or conversion to its alpha-anomer in 0.1 N HCI solution is not observed over a 4-week period. However, this work has shown that gemcitabine hydrochloride anomerizes in 0.1 N NaOH at 40 degrees C. Approximately 72% of the initial gemcitabine remains after 4 weeks under the basic conditions used. Uridine hydrolysis products are also formed under these conditions. The anormerization reaction, which is unusual under basic conditions, has been confirmed by characterization of the chromatographically isolated alpha-anomer by NMR and mass spectrometry. A mechanism involving an acyclic intermediate is proposed. 相似文献
88.
CC Helbing CL Wellington M Gogela-Spehar T Cheng GG Pinchbeck RN Johnston 《Canadian Metallurgical Quarterly》1998,17(12):1491-1501
When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states. 相似文献
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An 8-year-old boy presented with precocious puberty and a mediastinal mass. A computer search showed that this rare presentation is most common with germ cell tumor of the mediastinum in children with Klinefelter syndrome. The tumor was completely resected after preoperative chemotherapy, and the patient is well 2 years after the operation. In patients with Klinefelter syndrome, germ cell tumors are 50 times more common than in patients without Klinefelter syndrome, usually contain nonseminomatous elements, present at an earlier age, and are seldom testicular in location. 相似文献