Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.     

Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly.

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.     

Stable genetic transformation ofPicea mariana (black spruce) via particle bombardment

Stable genetic transformation ofPicea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell line were obtained from the mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014). The suspension culture from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they have a normal phenotype.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Recovery processes after repeated supramaximal exercise at the altitude of 4,350m

Robach, Paul, Daniel Biou, Jean-Pierre Herry, Denis Deberne,Murielle Letournel, Jenny Vaysse, and Jean-Paul Richalet. Recoveryprocesses after repeated supramaximal exercise at the altitude of 4,350 m. J. Appl. Physiol. 82(6):1897-1904, 1997.We tested the hypothesis that prolonged exposureto high altitude would impair the restoration of muscle power duringrepeated sprints. Seven subjects performed two 20-s Wingate tests (WT1and WT2) separated by 5 min of recovery, at sea level (N) and after5-6 days at 4,350 m (H). Mean power output (MPO) andO₂ deficit were measured duringWT. O₂ uptake(O₂) and ventilation(E) were measured continuously. Blood velocity in the femoral artery (FBV) wasrecorded by Doppler ultrasound during recovery. Arterialized blood pHand concentrations of bicarbonate([HCO₃]), venousplasma lactate([La]),norepinephrine ([NE]), and epinephrine ([Epi])were measured before and after WT1 and WT2. MPO decreased between WT1and WT2 by 6.9% in N (P < 0.05) andby 10.7% in H (P < 0.01). H did not further decrease MPO. O₂ deficitdecreased between WT1 and WT2 in H only(P < 0.01). PeakO₂ after WT was reduced by30-40% in H (P < 0.01), butexcess postexercise O₂ consumptionwas not significantly lowered in H. During recovery in H compared with N, E,exercise-induced acidosis, and [NE] were higher,[Epi] tended to be higher,[La] was notaltered, and [HCO₃] andFBV were lower. The similar[La]accumulation was associated with a higher exercise-induced acidosis anda larger increase in [NE] in H. We concluded from thisstudy that prolonged exposure to high altitude did not significantly impair the restoration of muscle power during repeated sprints, despitea limitation of aerobic processes during early recovery.

     

A plant in vitro system for the nuclear import of proteins

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-γ-S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP-γ-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.     

A 5.5-Mb High-Resolution Integrated Map of Distal 11q13

The distal part of 11q13, which contains several genes relevant to human diseases, has been poorly mapped as part of genome-wide mapping efforts. In the prospect of drawing a fine-scale integrated map of the area containingKRN1andOMP,we have established a framework of markers by hybridization to DNA of somatic cell hybrids and by fluorescencein situhybridization (FISH) on metaphase chromosomes. The probes studied were used to isolate 27 YACs and 16 cosmids that could be organized in three contigs covering approximately 6 Mb. These contigs were separated by two gaps that are likely to contain sequences underrepresented in YAC libraries. They were then integrated based on long-range restriction mapping and DNA-fiber FISH into a high-resolution physical map, which covers a 5.5-Mb region and includes 36 anonymous markers and 10 genes. This map will be used to search for genes within the 2/3 of this region where none have been localized as yet. It will also lay the ground for the characterization of an amplicon surroundingGARPin breast cancer and for the search of disease genes within this region.     

Biotin and choline replace the growth requirement of Madin-Darby canine kidney cells for high-density lipoproteins

MDCK cells maintained on extracellular matrix (ECM)-coated dishes and exposed to Dulbecco's modified Eagle's medium (DME) supplemented with transferrin and either high-density lipoproteins (HDLs) or phosphatidyl choline (PC) liposomes have a growth rate and final cell density similar to those of cultures exposed to serum-supplemented DME. When MDCK cells are exposed to a medium consisting of a mixture (1:1) of DME and F12 medium (D/F), the addition of transferrin (10 μg/ml) alone supports cell growth and the presence of HDLs or PC liposomes is no longer required. MDCK cells exposed to D/F medium supplemented with transferrin can be passaged for more than 50 generations in total absence of serum. The F12 components that support growth in the absence of HDLs or PC liposomes are biotin (which is absent in DME) and choline (which is present in insufficient concentration in DME). Supplementation of DME with transferrin, biotin (3.6 ng/ml), and choline (10 μg/ml) allows optimal growth of MDCK cells and permits serial propagation through more than 50 generations. The growth requirement of MDCK cells for HDLs or PC liposomes can therefore be replaced by adequate concentrations of biotin and choline. The widely observed fact that a combination of DME/F12 medium is more effective than DME alone in supporting cell growth may be due in part to the lack of biotin and suboptimal choline concentration in DME.     

On the separation of net ecosystem exchange into assimilation and ecosystem respiration: review and improved algorithm

This paper discusses the advantages and disadvantages of the different methods that separate net ecosystem exchange (NEE) into its major components, gross ecosystem carbon uptake (GEP) and ecosystem respiration (R_eco). In particular, we analyse the effect of the extrapolation of night‐time values of ecosystem respiration into the daytime; this is usually done with a temperature response function that is derived from long‐term data sets. For this analysis, we used 16 one‐year‐long data sets of carbon dioxide exchange measurements from European and US‐American eddy covariance networks. These sites span from the boreal to Mediterranean climates, and include deciduous and evergreen forest, scrubland and crop ecosystems. We show that the temperature sensitivity of R_eco, derived from long‐term (annual) data sets, does not reflect the short‐term temperature sensitivity that is effective when extrapolating from night‐ to daytime. Specifically, in summer active ecosystems the long‐term temperature sensitivity exceeds the short‐term sensitivity. Thus, in those ecosystems, the application of a long‐term temperature sensitivity to the extrapolation of respiration from night to day leads to a systematic overestimation of ecosystem respiration from half‐hourly to annual time‐scales, which can reach >25% for an annual budget and which consequently affects estimates of GEP. Conversely, in summer passive (Mediterranean) ecosystems, the long‐term temperature sensitivity is lower than the short‐term temperature sensitivity resulting in underestimation of annual sums of respiration. We introduce a new generic algorithm that derives a short‐term temperature sensitivity of R_eco from eddy covariance data that applies this to the extrapolation from night‐ to daytime, and that further performs a filling of data gaps that exploits both, the covariance between fluxes and meteorological drivers and the temporal structure of the fluxes. While this algorithm should give less biased estimates of GEP and R_eco, we discuss the remaining biases and recommend that eddy covariance measurements are still backed by ancillary flux measurements that can reduce the uncertainties inherent in the eddy covariance data.