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Asfari M, Thiébaud CH Cancer Res. 1988 Feb 15;48(4):954–7 The fate of the putative transplantable Xenopus lymphosarcoma (M. Balls, Cancer Res., 22: 1142-1154, 1962) was studied under three experimental conditions: (a) xenotransplantation, i.e., transplantation of live "tumor" tissue between adults of Xenopus borealis and X. laevis; (b) inoculation of live "tumor" cells from X. borealis into the blastocoele of X. laevis embryos; and (c) transplantation of "tumor" tissue into recipient adults immunologically unresponsive to the donor tissue antigens. This condition is fulfilled by using X. laevis-X. gilli (LG) hybrids [H.R. Kobel and L. Du Pasquier. In: J.B. Solomon and J.D. Horton (eds.), Developmental Immunology, pp. 229-306. The Netherlands: Elsevier/North-Holland Biomedical Press, 1977] as donors, and triploid X. laevis-X. gilli and X. borealis (LGB) hybrids [C.H. Thiébaud, Dev. Biol., 98: 245-249, 1983] as recipients. In all transplantationexperiments, donor and recipient cells could be unambiguously distinguished upon quinacrine staining that yields typical nuclear patterns, for instance bright patchiness in X. borealis, visible also in LGB cells. The results of xenotransplantation between X. borealis and X. laevis indicated that all developing "tumors" were composed of the recipient cell phenotype. The inoculation of live "tumor" cells from X. borealis "tumor" into the blastocoele of X. laevis embryos resulted in "tumor" formation in the recipient tadpoles and in metamorphosed animals. The cell constituting these "tumors" all were of recipient, X. laevis cell phenotype. Finally, "tumor" tissues from LG clones transplanted into LGB hosts were replaced by "tumors" formed of cells with recipient, LGB phenotype. These experiments indicate that this Xenopus tumor-like growth is a transmissible and not a transplantable disorder.

Bresler M, Behnam J, Luke G, Simkiss K Br Poult Sci. 1994 May;35(2):241–7 1. Embryos of the domestic fowl have been partially sterilised by injecting the drug busulphan into 24-h incubated eggs. 2. Some of these embryos were injected with primordial germ cells (PGCs) after 55 h of incubation to attempt to repopulate the gonads. 3. Primordial germ cells transfected with a defective retrovirus containing the reporter gene lac Z were shown to settle in these sterilised gonads. 4. Quantitative histology of 6-d embryos showed that busulphan produced 75% sterilisation but that PGCs could repopulate these gonads. 5. The technique of producing such germ line chimaeras is of value in studying cell kinetics, gonad differentiation and the production of transgenics.

Morita H, Yano Y, Niswender KD, May JM, Whitesell RR, Wu L, Printz RL, Granner DK, Magnuson MA, Powers AC J Clin Invest. 1994 Oct;94(4):1373–82 A Xenopus oocyte expression system was used to examine how glucose transporters (GLUT 2 and GLUT 3) and glucokinase (GK) activity affectglucose utilization. Uninjected oocytes and low rates of both glucose transport and phosphorylation; expression of GLUT 2 or GLUT 3 increasedglucose phosphorylation approximately 20-fold by a low Km, endogenous hexokinase at glucose concentrations < or = 1 mM, but not at higherglucose concentrations. Coexpression of functional GK isoforms with GLUT 2 or 3 increased glucose utilization approximately an additional two- to threefold primarily at the physiologic glucose concentrations of 5-20 mM. The Km for glucose of both the hepatic and beta cell isoforms of GK, determined in situ, was approximately 5-10 mM when coexpressed with either GLUT 2 or GLUT 3. The increase in glucose utilization bycoexpression of GLUT 3 and GK was dependent upon glucose phosphorylation since two missense GK mutations linked with maturity-onset diabetes, 182: Val-->Met and 228:Thr-->Met, did not increase glucose utilization despite accumulation of both a similar amount of immunoreactive GK protein and glucose inside the cell. Coexpression of a mutant GK and a normal GK isoform did not interfere with the function of the normal GK enzyme. Since the coexpression of GK and a glucose transporter in oocytes resembles conditions in the hepatocyte and pancreatic beta cell, these results indicate that increases in glucose utilization at glucose concentrations > 1 mM depend upon both a functional glucose transporter and GK.

Due AD, Qu Z-C, Thomas JM, Buchs A, Powers AC, May JM Biochemistry. ACS Publications; 1995;34(16):5462–71 Structural determinants for the glucose transport kinetics of the erythrocyte glucose transporter have not been established. In this work the role of the cytosolic carboxy-terminal tail in the expression and function of the human GLUT1 isoform in Xenopus oocytes was investigated. Oocyte plasma membrane expression of GLUT1 was a saturable function of the amount of mRNA injected. Transport activity increased as a linear function of the amount of immunoreactive transporter in the plasma membrane. Transport kinetics of human GLUT1 expressed in oocytes resembled those of human erythrocyte GLUT1. Addition of up to 31 extra amino acids to the carboxy-terminal tail of GLUT1 was without effect on its function in oocytes. Removal of the carboxy-terminal 21 amino acids also did not affect GLUT1 expression or transport kinetics in oocytes. Removal of the entire carboxy-terminal tail to Phe-450 resulted in a transporter that had moderately decreased plasma membrane expression compared to that of GLUT1. However, transport activity of this construct was less than 5% of that of GLUT1, and was associated with loss of its outward-facing inhibitor binding site. When the carboxy-terminal 29 amino acids of GLUT1 were replaced with the corresponding region of GLUT4, transporter expression in the plasma membrane and the transport Vmax fell to low levels, similar to those of native GLUT4. When the carboxy-terminal 29 or 73 amino acids ofGLUT1 were swapped into the corresponding region of GLUT4, the transport Vmax markedly increased to about one-third to one-half that of GLUT1, although the affinity for substrate was halved. These results show that the carboxy-terminal tail of the GLUT1 is not critical for targeting of the protein to the plasma membrane, but that this region is an important determinant of transport function.

Palma E, Mileo AM, Eusebi F, Miledi R Proc Natl Acad Sci USA. 1996 Oct 1;93(20):11231–5 A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

Gurley DA, Lanthorn TH Neurosci Lett. 1998 May 15;247(2-3):107-10 The 5-HT3 receptor (5-HT3R) is part of a superfamily of ligand-gated ion channels which includes nicotinic acetylcholine receptors (nAChR). cRNA derived from the long isoform cloned mouse 5-HT3R was used to drive expression of 5-HT3Rs in Xenopus oocytes. 5-HT-induced currents were monitored using two-electrode voltage-clamp. Eight nicotinic agonists, including ACh and nicotine, but not alpha-anatoxin, were found to antagonize 5-HT-induced currents. With the exception of 3-(2,4)-dimethoxybenzylidene-anabaseine (DMXB-anabaseine; GTS-21) this antagonism appeared to be competitive since it could be overcome by increasing concentrations of 5-HT. Potency of 5-HT3 antagonism was comparable to reported values for nAChR alpha7 activation. These results confirm the notion of families of receptors and further indicate that strong similarities can exist in some critical binding domains.

Wu L, Fritz JD, Powers AC Endocrinology. 1998 Oct;139(10):4205–12 GLUT2 is the major glucose transporter in pancreatic beta-cells and hepatocytes. It plays an important role in insulin secretion from beta-cells andglucose metabolism in hepatocytes. To better understand the molecular determinants for GLUT2's distinctive glucose affinity and its ability to transport fructose, we constructed a series of chimeric GLUT2/GLUT3 proteins and analyzed them in both Xenopus oocytes and mammalian cells. The results showed the following. 1) GLUT3/GLUT2 chimera containing a region from transmembrane segment 9 to part of the COOH-terminus ofGLUT2 had Km values for 3-O-methylglucose similar to those of wild-type GLUT2. Further narrowing of the GLUT2 component in the chimeric GLUTs lowered the Km values to those of wild-type GLUT3. 2) GLUT3/GLUT2 chimera containing a region from transmembrane segment 7 to part of the COOH-terminus of GLUT2 retained the ability to transport fructose. Further narrowing of this region in the chimeric GLUTs resulted in a complete loss of the fructose transport ability. 3) Chimeric GLUTs with the NH2-terminal portion of GLUT2 were unable to express glucose transporter proteins in either Xenopus oocytes or mammalian RIN 1046-38 cells. These results indicate that amino acid sequences in transmembrane segments 9-12 are primarily responsible for GLUT2's distinctive glucose affinity, whereas amino acid sequences in transmembrane segments 7-8 enable GLUT2 to transport fructose. In addition, certain region(s) of the amino-terminus of GLUT2 impose strict structural requirements on the carboxy-terminus of theglucose transporter protein. Interactions between these regions and the carboxy-terminus of GLUT2 are essential for GLUT2 expression.

Bailey CP, Dagle JM, Weeks DL Nucleic Acids Res. 1998 Nov 1;26(21):4860–7 Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selectiveinhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes.Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibitionof gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.

Bailey C, Weeks DL Nucleic Acids Res. 2000 Mar 1;28(5):1154–61 Triplex-forming oligonucleotides (TFOs) modified with N,N-diethylethylenediamine can inhibit the expression of a reporter plasmid in Xenopusoocytes if the triplex is preformed prior to injection while unmodified oligonucleotides cannot. Here we show that merely forming a triplex in a reporter plasmid does not disrupt transcription, but when TFOs are targeted to sites within the transcribed region of a reporter gene then gene activity is inhibited. TFO-based inhibition did not lead to large scale degradation or mutation of the reporter plasmid, but dramatically lowered mRNA levels. Finally, we investigated the accessibility of a triplex target site on a reporter plasmid after injection into nuclei. We found that the site used for our previous studies was inaccessible to restriction endonuclease after injection into nuclei. This observation may explain why inhibition was dependent on forming the triplex before injection into oocytes. Based on the assumption that oligonucleotide association, like restriction enzyme access, was excluded by nucleosome formation, additional target sites were inserted so that all sites could not simultaneously be associated with the octamer core of a nucleosome. With multiple target sites prior association of the plasmid with nuclear proteins does not prevent oligonucleotide-mediatedinhibition of gene activity.

Tanaka DH, Maekawa K, Yanagawa Y, Obata K, Murakami F Development. 2006 Jun;133(11):2167–76 Most GABAergic interneurons originate from the basal forebrain and migrate tangentially into the cortex. The migratory pathways and mode of interneuron migration within the developing cerebral cortex, however, previously was largely unknown. Time-lapse imaging and in vivo labelling with glutamate decarboxylase (GAD)67-green fluorescence protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)ergic neurons indicated that multidirectional tangential (MDT) migration of interneurons takes place in both the marginal zone (MZ) and the ventricular zone (VZ) of the cortex. Quantitative analysis of migrating interneurons showed that rostrocaudally migrating neurons outnumber those migrating mediolaterally in both of these zones. In vivo labelling with a lipophilic dye showed that the MDT migration in the MZ occurs throughout thecortex over distances of up to 3 mm during a period of a few days. These results indicate that MZ cortical interneurons undergo a second phase oftangential migration in all directions and over long distances, after reaching the cortex by dorsomedial tangential migration. The MDT migration in the MZ may disperse and intermix interneurons within the cortex, resulting in a balanced distribution of interneuron subtypes.