Widely Used Enhancer of Eukaryotic Expression Vectors Is Strongly and Differentially Regulated in Fibroblast, Myoblast, and Teratocarcinoma Cell Lines

The expression of transgenes in eukaryotic cells is a powerful approach in cell biology. In most cases, it is based on the activity of strong and constitutive viral cis-acting elements in eukaryotic expression vectors. Here we show that a widely used such element derived from an early gene of human cytomegalovirus is strongly and differentially regulated in mouse cell lines. We analyzed cytomegalovirus promoter-driven expression of stably transfected transgenes in growing, confluent, and differentiating mouse 3T3 fibroblasts, C2C12 myoblasts, and P19 teratocarcinoma cells. In the fibroblasts, transgene expression was strongly downregulated in confluent cultures and was upregulated in growing or confluent cultures by phorbol ester. In contrast, no downregulation by confluency, nor upregulation by phorbol ester, was detected in C2C12 cells. In addition, while marked upregulation was detected in differentiating myotubes, transgene expression was downregulated when differentiating teratocarcinoma cells assumed a neuronal phenotype. These results demonstrate the existence of drastic differences in the regulation of transgene expression in different types of cell lines, indicating that when studying transgene function in cells that are not growing exponentially, viral promoter-driven expression should not be taken for granted.     

High-resolution mapping of a linkage group on mouse chromosome 8 conserved on human chromosome 16Q

We have performed a high-resolution linkage analysis for the conserved segment on distal mouse Chromosome (Chr) 8 that is homologous to human Chr 16q. The interspecific backcross used involved M. m. molossinus and an M. m. domesticus line congenic for an M. spretus segment from Chr 8 flanked by phenotypic markers Os (oligosyndactyly) and e, a coat colormarker. From a total of 682 N₂ progeny, the 191 animals revealing a recombination event between these phenotypic markers were typed for 23 internal loci. The following locus order with distances in cM was obtained: (centromere)–Os–4.1–Mmp2–0.2–Ces1,Es1, Es22–1.2–Mt1,D8Mit15–2.2–Got2, D8Mit11–3.7–Es30–0.3–Es2, Es7–0.9–Ctra1,Lcat–0.3–Cdh1, Cadp, Nmor1, D8Mit12–0.2–Mov34–2.5–Hp,Tat–0.2–Zfp4–1.6–Zfp1,Ctrb–10.9–e. In a separate interspecific cross involving 62 meioses, Dpep1 was mapped together with Aprt and Cdh3 at 12.9 cM distal to Hp, Tat, to the vicinity of e. Our data give locus order for markers not previously resolved, add Mmp2 and Dpep1 as new markers on mouse Chr 8, and indicate that Ctra1 is the mouse homolog for human CTRL. Comparison of the order of 17 mouse loci with that of their human homologs reveals that locus order is well conserved and that the conserved segment in the human apparently spans the whole long arm of Chr 16. Received: 30 July 1996 / Accepted: 15 November 1996     

Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

TRPC3 (or Htrp3) is a human member of the trp family of Ca²⁺-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca²⁺ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca²⁺ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca²⁺ over Na⁺. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca²⁺ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca²⁺. Likewise, infusion of Ca²⁺ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca²⁺-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca²⁺ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca²⁺-permeable channel that supports Ca²⁺-induced Ca²⁺-entry but should not be considered store operated.     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs

Theodorou, Andreas, Natalie Weger, Kathleen Kunke, KyooRhee, David Bice, Bruce Muggenberg, and Richard Lemen. Ragweed sensitization alters pulmonary vascular responses to bronchoprovocation in beagle dogs. J. Appl. Physiol.83(3): 912-917, 1997.In ragweed (RW)-sensitized beagle dogs, wetested the hypothesis that reactivity of the pulmonary vasculature wasenhanced with aerosolized histamine (Hist) and RW. Seven dogs wereneonatally sensitized with repeated intraperitoneal RW injections, and12 dogs were controls (Con). The dogs were anesthetizedwith intravenous chloralose, mechanically ventilated, and instrumentedwith femoral arterial and pulmonary artery catheters. Specific lungcompliance(CL_sp),specific lung conductance (G_sp),systemic vascular resistance index, and pulmonary vascular resistanceindex (PVRI) were measured before and after bronchoprovocation withHist and RW. After Hist inhalation (5 breaths of 30 mg/ml), both Conand RW dogs had significant (P < 0.05) decreases inCL_sp(51 ± 4 and 53 ± 5%, respectively) andG_sp (65 ± 5 and69 ± 3%, respectively), but only RW-sensitized dogs had asignificant increase in PVRI (38 ± 10%). After RW inhalation (60 breaths of 0.8 mg/ml), only RW-sensitized dogs had significant increases (62 ± 20%) in PVRI and decreases inG_sp (77 ± 4%) and CL_sp(65 ± 7%). We conclude that, compared with Con,RW-sensitized beagle dogs have increased pulmonary vasoconstrictiveresponses with Hist or RW inhalation.

     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.