Antisera inhibiting mammalian cell spreading and possible cell surface antigens involved.

Antiserum against a rat neuronal tumor cell line (B103) has been prepared in rabbit by intravenous injection of live cells. This immune serum (anti-B103) precipitates a few cell surface proteins recognizable by two-dimensional gel electrophoresis as common radioiodinatable spots in 15 different rat neural cell lines and in mouse and rat fibroblast cell lines. The apparent molecular weight of one major common protein (II4) is estimated by SDS gel electrophoresis to be somewhere between 80,000 and 90,000 and another protein (I3) to be 120,000. These two proteins are consistently recognized in various cell lines by this antiserum. Furthermore, at a 1:20 dilution, this serum causes monolayer cells to round up usually within 0.5 h and detach from the plate within 3 h. It also inhibits spreading of freshly plated cells. These effects of the antiserum are reversible. Upon absorption of the antiserum with cells (e.g., absorbed with a glial cell line, B27), the serum no longer causes the rounding up of the monolayer cells, it does not inhibit cell spreading, and it does not immune-precipitate the two common proteins from the cell surface of various cell lines. Antisera against several other rat cell lines also precipitate the same common proteins (II4 and I3) from the cell surface and prevent cell spreading. These data suggest that the antibody acts first at the cell surface and then inhibits cell spreading or rounding up of spread cells. The consistent pattern of the immunoprecipitated cell surface proteins on the two-dimensional gel electrophoresis makes these two common surface proteins (II4 or I3 or both) possible candidates for target proteins to which the antibody binds. Thus, they may play a critical role in cell spreading.

Cloning of DNA corresponding to rare transcripts of rat brain: evidence of transcriptional and post-transcriptional control and of the existence of nonpolyadenylated transcripts.

To examine the expression of genes encoding rare transcripts in the rat brain, we have characterized genomic DNA clones corresponding to this class. In brain cells, as in all cell types, rare transcripts constitute the majority of different sequences transcribed. Moreover, when compared with other tissues or cultured cells, brain tissue may be expected to have an even larger set of rare transcripts, some of which could be restricted to subpopulations of neural cells. We have identified seven clones whose transcripts are nonabundant, averaging less than three copies per cell. Clone rg13 (rat genomic 13) RNA was detected only in the brain, whereas RNA of a second clone, rg40, was also detected in the brain and in a melanoma. Transcripts of rg13 were found in cerebellum, cerebral cortex, and regions underlying the cortex, whereas rg40 transcripts were not detected in the cerebellum. Transcripts of both rg13 and rg40 were found in pelleted polysomal RNA. RNA of another clone, rg34, was found in the brain, liver, and kidney but was found in pelleted polysomal RNA only in the brain, suggesting that its expression may be post-transcriptionally controlled. The remaining four clones represent rare transcripts that are common to the brain, liver, and kidney; rg18 RNA is restricted to the nucleus, whereas rg3, rg26, and rg36 transcripts are found in the cytoplasm of all three tissues. Transcripts of the brain-specific clone, rg13, and the commonly expressed clone, rg3, are nonpolyadenylated, presumably belonging to the high-complexity, nonpolyadenylated class of transcripts in the mammalian brain.

All-trans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity.

Jun N-terminal kinases (JNKs) are serine-threonine kinases that play a critical role in the regulation of cell growth and differentiation. We previously observed that JNK activity is suppressed by all-trans-retinoic acid (t-RA), a ligand for retinoic acid nuclear receptors (RARs), in normal human bronchial epithelial cells, which are growth inhibited by t-RA. In this study, we investigated the mechanism by which t-RA inhibits JNK and the possibility that this signaling event is blocked in non-small cell lung cancer (NSCLC) cells. Virtually all NSCLC cell lines are resistant to the growth-inhibitory effects of t-RA, and a subset of them have a transcriptional defect specific to retinoid nuclear receptors. We found that in NSCLC cells expressing functional retinoid receptors, serum-induced JNK phosphorylation and activity were inhibited by t-RA in a bimodal pattern, transiently within 30 min and in a sustained fashion beginning at 12 h. Retinoid receptor transcriptional activation was required for the late, but not the early, suppression of JNK activity. t-RA inhibited serum-induced JNK activity by blocking mitogen-activated protein (MAP) kinase kinase 4-induced signaling events. This effect of t-RA was phosphatase dependent and involved an increase in the expression of the dual-specificity MAP kinase phosphatase 1 (MKP-1). t-RA did not activate MKP-1 expression or inhibit JNK activity in a NSCLC cell line with retinoid receptors that are refractory to ligand-induced transcriptional activation. These findings provide the first evidence that t-RA suppresses JNK activity by inhibiting JNK phosphorylation. Retinoid receptor transcriptional activation was necessary for the sustained inhibition of JNK activity by t-RA, and this signaling event was disrupted in NSCLC cells with retinoid receptors that are refractory to ligand-induced transcriptional activation.

Cell membrane and chromosome replication in Bacillus subtilis.

This review covers studies of the structural and functional roles of the cell membrane on the replication of the Bacillus subtillis chromosome. A particular emphasis is placed on the essential roles of the membrane complex for the in vivo initiation and termination of the chromosome replication. A critical gene complex in B. subtillis for the role of membrane complex is the dnaB operon that most likely consists of four genes (dnaB, dnaI, ORFZ/ORF213, and ORF omega/ORF281). Detailed studies of these genes are currently available only for the dnaB and dnaI genes. The unique feature of the dnaB gene is that temperature-sensitive mutants of this gene simultaneously lose, at the nonpermissive temperature, chromosome attachment at oriC to the membrane as well as the new round of replication initiation at oriC. Further studies on the genes and their products of the dnaB operon are therefore essential for our understanding of the in vivo mechanism of the initiation of chromosome replication and its regulation. The role of the membrane on the termination and segregation of the daughter chromosomes has not been discovered, but an important clue comes from the terminus area of the B. subtillis chromosome being bound to the membrane in a high-salt resistant and DnaB-independent fashion.

Heterogeneous nuclear ribonucleoprotein B1 as a new marker of early detection for human lung cancers.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an RNA binding protein that is required for maturation of mRNA precursor. Tockman et al. previously reported that hnRNP A2/B1 with a M(r) of 31,000 is overexpressed from the early clinical stage of human lung cancer (M. S. Tockman et al., J. Clin. Oncol., 6: 1685-1693, 1988). However, when hnRNP A2/B1 mRNA and hnRNP B1 mRNA were separately studied, we found unique evidence that hnRNP B1 mRNA, which is a splicing variant of hnRNP A2 mRNA, was more significantly elevated in lung cancer tissues than hnRNP A2/B1 mRNA. Our hnRNP B1-specific polyclonal antibody specifically recognized hnRNP B1 protein as a M(r) 37,000 nuclear protein by Western blotting but did not recognize hnRNP A2 protein. Immunohistochemical staining with the hnRNP B1 antibody revealed that hnRNP B1 protein was specifically stained in the nuclei of human cancer cells, and in squamous cell carcinomas in particular, but not in those of normal adjacent lung epithelial cells. We think that hnRNP B1 protein of M(r) 37,000, not hnRNP A2, is well qualified as a biomarker for the detection of human lung cancer.

Synergistic effects of (--)-epigallocatechin gallate with (--)-epicatechin, sulindac, or tamoxifen on cancer-preventive activity in the human lung cancer cell line PC-9.

The study on incorporation of [3H](-)-epigallocatechin gallate (EGCG) into human lung cancer cell line PC-9 indicated that the [3H]EGCG incorporation was significantly enhanced by (-)-epicatechin, an inert tea polyphenol without a galloyl moiety. (-)-Epicatechin enhanced apoptosis, growth inhibition of PC-9 cells, and inhibition of tumor necrosis factor-alpha release from BALB/c-3T3 cells by EGCG and other tea polyphenols with a galloyl moiety in a dose-dependent manner. Moreover, the effects of EGCG on induction of apoptosis were also synergistically enhanced by other cancer-preventive agents, such as sulindac and tamoxifen. This paper reports significant evidence that whole green tea is a more reasonable mixture of tea polyphenols for cancer prevention in humans than EGCG alone and that it is even more effective when it is used in combination with other cancer preventives.

Posttranslational mechanisms contribute to the suppression of specific cyclin:CDK complexes by all-trans retinoic acid in human bronchial epithelial cells.

Retinoids have demonstrated activity in the chemoprevention of aerodigestive tract cancer. Potentially contributing to their lung cancer chemopreventive effects, retinoids inhibit the growth of human bronchial epithelial (HBE) cells. We observed previously that all-trans retinoic acid (t-RA) arrests the growth of HBE cells in the G0 phase of the cell cycle through activation of retinoic acid receptor-dependent pathways, which enhances the association of E2F-4 with retinoblastoma protein family members, converting E2F into a transcriptional suppressor. In this study, we examined the mechanism by which t-RA blocks cell cycle progression in HBE cells and the possibility that this signaling event is blocked in non-small cell lung cancer (NSCLC) cells that are refractory to the growth inhibitory effects of t-RA. t-RA suppressed the expression and activity of cyclin D1, cyclin E, and cyclin-dependent kinases (CDK)-2 and CDK-4, increased expression of the CDK inhibitor p27, and shifted the retinoblastoma protein to a hypophosphorylated form. Posttranslational mechanisms contributed to the changes in CDK-2, CDK-4, and p27 levels, which, in the case of CDK-4, involved the ubiquitin-proteasome pathway. In contrast, despite retinoic acid receptor transcriptional activation, these signaling events did not occur in a NSCLC cell line that is refractory to growth inhibition by t-RA. These findings provide the first evidence that t-RA activates degradation of CDK-4 through the ubiquitin-proteasome pathway, a novel mechanism by which t-RA causes HBE cells to exit the cell cycle, and blockade of these signaling events may contribute to the development of retinoid resistance in NSCLC cells.

Helicobacter pylori membrane protein 1: a new carcinogenic factor of Helicobacter pylori.

Considering a suspected link between Helicobacter pylori infection and human stomach cancer, a new H. pylori gene for membrane protein 1 (HP-MP1) was recently cloned. Because HP-MP1 induces release of inflammatory cytokines and tumor necrosis factor-alpha acts as both initiator and tumor promoter, we studied the possible involvement of HP-MP1 in carcinogenesis of H. pylori. Two cell lines, BALB/3T3 cells as control and v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells) as putative initiated cells, were each transfected with HP-MP1, urease B genes, or vector alone. All of the Bhas/mpl clones showed strong expression of tumor necrosis factor-alpha gene and produced tumors in 100% of nude mice. Two Bhas/ure clones showed weak tumorigenicity; the other Bhas and BALB clones showed none. Results indicate strong carcinogenic activity of HP-MP1 in cooperation with viral Ras protein and weak activity of urease B.

Heterogeneous nuclear ribonucleoprotein B1 as early cancer biomarker for occult cancer of human lungs and bronchial dysplasia.

Heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is a RNA-binding protein of Mr 37,000. We previously reported that hnRNP B1 was specifically overexpressed in the nuclei of human lung cancer cells, particularly in squamous cell carcinoma (E. Sueoka et al., Cancer Res., 59: 1404-1407, 1999). We extended this study to determine whether hnRNP BL was overexpressed in roentgenographically occult cancers of the lungs and premalignant lesions of squamous cell carcinomas, such as bronchial dysplasia. The additional object of our study was to examine the usefulness of hnRNP B1 as a potential diagnostic marker for squamous cell carcinoma of various organs, such as the oral cavity and esophagus in humans. Surgically resected specimens of bronchial dysplasia, lung cancers, and various human squamous cell carcinomas, collected at two hospitals in Japan, were subjected to immunohistochemical staining with anti-hnRNP B1 antibody. Overexpression of hnRNP B1 protein was observed in 100% of stage I lung cancer tissues, but it was not found in normal bronchial epithelium. Squamous cell carcinoma of the lungs showed stronger staining than other histological types, and elevation of hnRNP B1 was found in both roentgenographically occult lung cancers and bronchial dysplasia. Furthermore, cytological examination with anti-hnRNP B1 antibody detected cancer cells in sputum, suggesting the potential of hnRNP B1 protein as a new biomarker for the very early stage of lung cancer in humans. Because strong staining of hnRNP B1 was also observed in various squamous cell carcinomas of oral and esophageal tissues as shown in our recent reports, overexpression of hnRNP B1 seems to be a common event in the carcinogenic processes of squamous cell carcinoma. These results suggest that hnRNP B1 protein could be a useful diagnostic biomarker for both the very early stages of lung cancer and various squamous cell carcinomas in humans.

Insulin-like growth factor binding protein-6 activates programmed cell death in non-small cell lung cancer cells.

Insulin-like growth factor binding proteins (IGFBPs) are secreted into the extra-cellular matrix and inhibit cell growth through IGF-dependent and -independent mechanisms. In this study, we investigated the role of IGFBP-6, a relatively unexplored member of the IGFBP family, in the proliferation of non-small cell lung cancer (NSCLC) cells. Infection of NSCLC cell lines in vitro with an adenovirus expressing human IGFBP-6 under the control of a CMV promoter (Ad5CMV-BP6) reduced NSCLC cell number through activation of programmed cell death, as shown by cell staining with Hoechst 33342 or DNA end-labeling with bromodeoxyuridine triphosphate. The growth regulatory effect of IGFBP-6 was investigated in vivo by intratumoral injection of Ad5CMV-BP6 in NSCLC xenografts established in nu/nu mice. A single injection of Ad5CMV-BP6 reduced the size of NSCLC xenografts by 45%. These findings indicate that IGFBP-6 is a potent inducer of programmed cell death in cancer cells and support investigations into IGFBP-6 as a potential target in cancer therapeutics.

A two-dimensional polyacrylamide gel electrophoresis system for the analysis of mammalian cell surface proteins.

A detailed analysis of mammalian cell surface proteins is described by a new two-dimensional polyacrylamide gel electrophoresis technique. The first dimension gel contains 2% acrylamide, 0.1% sodium dodecyl sulfate, 0.3% Triton CF10 and 9 M urea. A combination of the detergents and urea permits the separation of poorly soluble, hydrophobic cell surface proteins. Under these conditions, the molecular size of proteins has a limited contribution to the fianl separation due to a low acrylamide concentration. Differences in charge properties, hydrophobicity, and glycosylation are the elements determining the resolution. In the second dimension, the proteins are separated primarily according to molecular weights, by a conventional polyacrylamide gel system in the presence of 0.1% sodium dodecyl sulfate. In this study, proteins of C6 rat glioma cell line are characterized. Cell surface proteins are specifically radio-labeled with 125I by a lactoperoxidase method, and compared with presumptive integral surface proteins which are resistant to extraction with 0.1 M NaOH. Also studied are total cellular proteins, fucose- and glucosamine-containing glycoproteins, and protein species with variable susceptibility to weak trypsin digestion. The electrophoresis system allows an unambiguous identification of each protein species.

Temperature-sensitive DNA synthesis mutants of Bacillus subtilis--appendix: theory of density transfer for symmetric chromosome replication.

A simple method for characterizing temperature-sensitive DNA synthesis mutants is described. The method uses density transfer and transformation techniques and is based on expected theoretical behavior of the chromosome population. A direct proof of inhibition of initiation of DNA replication is provided. The mutant dna-1, showing quick inhibition of initiation, is further characterized and mapped. An independent method for mapping genetic markers close to the origin, based on their transfer behavior after inhibition of initiation, is presented.

Genetic mapping of a group of temperature-sensitive dna initiation mutants in Bacillus subtilis.

Recombination frequencies among temperature-sensitive dna mutants from various laboratories were analyzed, and eleven dna mutants were found to be closely linked. They are classified as group B dna mutants, since these are closely linked with dnaB19, originally isolated and approximately mapped near leuA8 by KARAMATA and GROSS (1970). However, the dnaB19 mutation itself has relatively high recombination frequencies with the other mutations, thus, we propose to subdivide the dnaB group into two subgroups--dnaBI, including ten mutants (dna-1, dna-3, dna-5, dna-17, dna-27, dna-51, dna-60, dna-62, dna-103 and dna-134) and dnaBII, including dnaB19. The map order of dnaB and markers in the vicinity was determined to be argA-citH-citC-phoP-PhoR-polA-dnaBI-dnaBII-citF-leuA-pheA.

Randomized study of orally administered fluorinated pyrimidines (capecitabine versus S-1) in women with metastatic or recurrent breast cancer: Japan Breast Cancer Research Network 05 Trial.

PURPOSE: Capecitabine and S-1 are orally administered fluorinated pyrimidines with high-level activity against metastatic breast cancer (MBC). This randomized, multicenter, phase II study compared the activities and safeties of the oral fluoropyrimidines, capecitabine and S-1, in breast cancer patients. METHODS: Patients with MBC were randomly assigned to receive capecitabine 825 g/m(2) twice daily on days 1-21 every 4 weeks or S-1 40-60 mg twice daily, according to body surface area, on days 1-28 every 6 weeks. The primary endpoint was progression-free survival (PFS). RESULTS: A total of 142 patients were enrolled and randomized to either capecitabine (N = 73) or S-1 (N = 69). Median PFS (progression-free survival) was 1.2 years for capecitabine and 1.3 years for S-1, with a hazard ratio (S-1/capecitabine) of 0.85 (95 % confidence interval [CI] 0.52-1.38) (P = 0.48 by log-rank). The confirmed objective response rates were 24.0 % for capecitabine and 23.1 % for S-1 (P = 0.938). The most common treatment-related adverse events were grade 1-2 in intensity. Thrombocytopenia (S-1: 9.2 %, capecitabine: 1.4 %; P = 0.040) and nausea (S-1: 26.2 %, capecitabine: 14.1 %; P = 0.079) were more frequent in the S-1 group, while hand-foot syndrome occurred more often in the capecitabine group (S-1: 10.8 %, capecitabine: 25.4 %; P = 0.029). CONCLUSIONS: The results of the current study demonstrate that both S-1 and capecitabine are effective and well-tolerated treatments in patients with MBC, while their adverse events were different. They are both convenient, orally administered drugs, making them attractive agents for use in outpatient treatment.

Translation-coupled violation of Parity Rule 2 in human genes is not the cause of heterogeneity of the DNA G+C content of third codon position.

The genome of higher eukaryotes consists of genes having a widely heterogeneous base composition at the third codon position. Ubiquitous variability of the DNA base composition has the following two aspects: intragenomic heterogeneity of the G+C content and the amino-acid-specific translation-coupled biases from the Parity Rule 2 (PR2). PR2 is an intrastrand rule where A = T and G = C are expected if there is no bias in mutation and selection between the two complementary strands of DNA. To examine whether or not the biases from PR2 are responsible for the wide heterogeneity of the DNA G+C content in human, the third codon position of 846 human genes was analyzed. Genes were separated into six groups according to their G+C content of the third codon position, and each group was examined for the translation-coupled PR2 biases in the nucleotide composition of the third codon position for two- and four-codon amino acids. The results show that genes in the different G+C content groups have similar PR2 biases, indicating that the intragenomic heterogeneity of the G+C content is not correlated with translation-coupled biases from the PR2. Therefore, the heterogeneity of the G+C content is likely to be determined by some other mechanism (e.g. locally variable directional mutation pressures) than amino-acid-specific selections for the codon preference.

DNA G+C content of the third codon position and codon usage biases of human genes.

The human genome, as in other eukaryotes, has a wide heterogeneity in the DNA base composition. The evolutionary basis for this heterogeneity has been unknown. A previous study of the human genome (846 genes analyzed) has shown that, in the major range of the G+C content in the third codon position (0.25-0.75), biases from the Parity Rule 2 (PR2) among the synonymous codons of the four-codon amino acids are similar except in the highest G+C range (Sueoka, N., 1999. Translation-coupled violation of Parity Rule 2 in human genes is not the cause of heterogeneity of the DNA G+C content of third codon position. Gene 238, 53-58.). PR2 is an intra-strand rule where A=T and G=C are expected when there are no biases between the two complementary strands of DNA in mutation and selection rates (substitution rates). In this study, 14,026 human genes were analyzed. In addition, the third codon positions of two-codon amino acids were analyzed. New results show the following: (a) The G+C contents of the third codon position of human genes are scattered in the G+C range of 0.22-0.96 in the third codon position. (b) The PR2 biases are similar in the range of 0.25-0.75, whereas, in the high G+C range (0.75-0.96; 13% of the genes), the PR2-bias fingerprints are different from those of the major range. (c) Unlike the PR2 biases, the G+C contents of the third codon position for both four-codon and two-codon amino acids are all correlated almost perfectly with the G+C content of the third codon position over the total G+C ranges. These results support the notion that the directional mutation pressure, rather than the directional selection pressure, is mainly responsible for the heterogeneity of the G+C content of the third codon position.

Selection of rat genomic clones transcribed into brain polysomal RNA.

A library of rat DNA in the lambda phage vector Charon 4A was screened with a cDNA probe transcribed from a brain polysomal poly(A)+RNA template. Clones were selected that served as templates for transcripts which were more abundant in brain than in liver. It was essential to add a large excess of repetitive DNA to the hybridization mixtures to prevent the cDNA from annealing to ubiquitous repetitive rat DNA in the clones. The abundance of the brain and liver polysomal poly(A)+ transcripts of one clone was determined.

Intra-strand biases in bacteriophage T4 genome.

In bacteriophage T4, a major portion of DNA replication is initiated at random along the map, although several proven and putative origins have been described for early replication. In order to analyze the contribution of transcription and translation as well as DNA replication to intra-strand bias from A = T and G = C, we examined the pattern of the intra-strand biases in the first, second, and third codon positions of the coding regions as well as the intergenic regions of the T4 genome. We found, along the map, characteristic biases both from A = T and G = C for each codon position and the intergenic regions. The bias patterns were closely associated with the location of the sense and anti-sense segments in the genome. The results suggest that: (1) transcription-associated mutation is likely a significant cause of the bias, which is suggested by the pattern of the AT bias (bias from A = T) in the third codon position; (2) DNA replication coupled bias may also exist, which is suggested by the pattern of the GC bias (bias from G = C) in the third codon position and the intergenic regions; and (3) the bias patterns of the first and second codon positions of the sense segments are consistent with universal properties of the coding sequence that G is in excess and T is deficient in the first codon position, and G is deficient in the second codon position.

In vitro catalytic activities of DNA/RNA chimeric hammerhead ribozymes against AML1-MTG8 mRNA, a fused gene transcript in acute myeloid leukemia with t(8;21).

In order to design the best construct for therapeutic hammerhead ribozymes against AML1-MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia, we synthesized DNA/RNA chimeric ribozymes directed to the area adjacent to the fusion point between AML1 and MTG8. Catalytic efficiency and fusion gene specificity of ribozymes were examined by kinetic studies of the cleavage reactions of AML1-MTG8, AML1, and MTG8 RNAs transcribed in vitro. Ribozyme 2 (Rz2) specifically cleaved AML1-MTG8 RNA at three nucleotides downstream of the fusion junction with high efficiency. The highest cleavage efficiency was achieved by Rz4.3, which targeted non-contiguous sequences and cleaved at 19 nucleotides downstream of the fusion junction. Rz4.3 also cleaved MTG8 RNA but the cleavage efficiency was three orders of magnitude lower than that for AML1-MTG8 RNA. Therefore, Rz4.3 and Rz2 are the proper ribozymes for in vivo application to modulate gene expression of the AML1-MTG8.

Characterization of transgenic mice expressing apolipoprotein E4(C112R) and apolipoprotein E4(L28P; C112R).

Apolipoprotein E (ApoE), which is genetically polymorphic, is a constituent of different lipoproteins. Two variants, ApoE4(C112R) and ApoE4(L28P; C112R) have been linked to the risk of developing Alzheimer's disease. Transgenic mice carrying ApoE4(C112R) (AD71) and ApoE4(L28P; C112R) (AD61) were generated and compared to wild-type mice. The use of glial fibrillary acidic protein as promoter led to transgene expression mainly in glial cells but also in neurons. Transgene protein levels were approximately three-and-a-half-fold that of endogenous ApoE in the glial fibrillary acidic protein-ApoE4(C112R) (AD71) and nearly twofold in the glial fibrillary acidic protein-ApoE4(L28P; C112R) (AD61) mouse lines. Neither transgenic mouse differed from wild-type in cognitive tests at the age of approximately one-and-a-half years. The locomotor activity of AD61 mice was similar to controls, whereas AD71 mice exhibited a clearly reduced level of motor activity. Immunohistological and biochemical brain protein analyses revealed no difference between strains.Thus, in the absence of morphological changes over-expression of ApoE4(C112R) on a background of endogenous mouse ApoE, may result in behavioral deficits while for the ApoE4(L28P; C112R) transgene higher expression might be required or some compensatory mechanisms might protect these animals from the behavioral abnormalities.