Immune regulation by the ST6Gal sialyltransferase

The ST6Gal sialyltransferase controls production of the Siaα2-6Galβ1-4GlcNAc (Sia6LacNAc) trisaccharide, which is the ligand for the lectin CD22. Binding of CD22 to Sia6LacNAc is implicated in regulating lymphocyte adhesion and activation. We have investigated mice that lack ST6Gal and report that they are viable, yet exhibit hallmarks of severe immunosuppression unlike CD22-deficient mice. Notably, Sia6LacNAc-deficient mice display reduced serum IgM levels, impaired B cell proliferation in response to IgM and CD40 crosslinking, and attenuated antibody production to T-independent and T-dependent antigens. Deficiency of ST6Gal was further found to alter phosphotyrosine accumulation during signal transduction from the B lymphocyte antigen receptor. These studies reveal that the ST6Gal sialyltransferase and corresponding production of the Sia6LacNAc oligosaccharide are essential in promoting B lymphocyte activation and immune function.

Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice

Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor l-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug d-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.

Inhibition of the electrostatic interaction between β-amyloid peptide and membranes prevents β-amyloid-induced toxicity

The accumulation of β-amyloid peptides (Aβ) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Aβ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Aβ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Aβ1-42 and the toxic fragment Aβ25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic β-sheet conformation by Aβ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Aβ1-40 and Aβ25-35 to negatively charged lipid vesicles and Aβ induced cell toxicity. These results suggest that Aβ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.

Apolipoprotein E is essential for amyloid deposition in the APPV717F transgenic mouse model of Alzheimer's disease

We quantified the amount of amyloid β-peptide (Aβ) immunoreactivity as well as amyloid deposits in a large cohort of transgenic mice overexpressing the V717F human amyloid precursor protein (APPV717F+/− TG mice) with no, one, or two mouse apolipoprotein E (Apoe) alleles at various ages. Remarkably, no amyloid deposits were found in any brain region of APPV717F+/− Apoe−/− TG mice as old as 22 mo of age, whereas age-matched APPV717F +/− Apoe+/− and Apoe+/+ TG mice display abundant amyloid deposition. The amount of Aβ immunoreactivity in the hippocampus was also markedly reduced in an Apoe gene dose-dependent manner (Apoe+/+ > Apoe+/− ≫ Apoe−/−), and no Aβ immunoreactivity was detected in the cerebral cortex of APPV717F+/− Apoe−/− TG mice at any of the time points examined. The absence of apolipoprotein E protein (apoE) dramatically reduced the amount of both Aβ1–40 and Aβ1–42 immunoreactive deposits as well as the resulting astrogliosis and microgliosis normally observed in APPV717F TG mice. ApoE immunoreactivity was detected in a subset of Aβ immunoreactive deposits and in virtually all thioflavine-S-fluorescent amyloid deposits. Because the absence of apoE alters neither the transcription or translation of the APPV717F transgene nor its processing to Aβ peptide(s), we postulate that apoE promotes both the deposition and fibrillization of Aβ, ultimately affecting clearance of protease-resistant Aβ/apoE aggregates. ApoE appears to play an essential role in amyloid deposition in brain, one of the neuropathological hallmarks of Alzheimer's disease.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Transforming neural computations and representing time

Motifs of neural circuitry seem surprisingly conserved over different areas of neocortex or of paleocortex, while performing quite different sensory processing tasks. This apparent paradox may be resolved by the fact that seemingly different problems in sensory information processing are related by transformations (changes of variables) that convert one problem into another. The same basic algorithm that is appropriate to the recognition of a known odor quality, independent of the strength of the odor, can be used to recognize a vocalization (e.g., a spoken syllable), independent of whether it is spoken quickly or slowly. To convert one problem into the other, a new representation of time sequences is needed. The time that has elapsed since a recent event must be represented in neural activity. The electrophysiological hallmarks of cells that are involved in generating such a representation of time are discussed. The anatomical relationships between olfactory and auditory pathways suggest relevant experiments. The neurophysiological mechanism for the psychophysical logarithmic encoding of time duration would be of direct use for interconverting olfactory and auditory processing problems. Such reuse of old algorithms in new settings and representations is related to the way that evolution develops new biochemistry.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9–11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-d-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not γ-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

Activation of iron regulatory protein-1 by oxidative stress in vitro

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-γS and GTP-γS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Identification and dissection of an enhancer controlling epithelial gene expression in skin

Keratins 14 and 5 are the structural hallmarks of the basal keratinocytes of the epidermis and outer root sheath (ORS) of the hair follicle. Their genes are controlled in a tissue-specific manner and thus serve as useful tools to elucidate the regulatory mechanisms involved in keratinocyte-specific transcription. Previously we identified several keratinocyte-specific DNase I hypersensitive sites (HSs) in the 5′ regulatory sequences of the K14 gene and showed that a 700-bp regulatory domain encompassing HSs II and III can confer epidermal and ORS-specific gene expression in transgenic mice in vivo. Although HS II harbored much of the transactivation activity in vitro, it was not sufficient to restrict expression to keratinocytes in vivo. We now explore the HS III regulatory element. Surprisingly, this element on its own confers gene expression to the keratinocytes of the inner root sheath (IRS) of the hair follicle, whereas a 275-bp DNA fragment containing both HSs II and III shifts the expression from the IRS to the basal keratinocytes and ORS in vivo. Electrophoretic mobility-shift assays and mutational studies of HSs III reveal a role for CACCC-box binding proteins, Sp1 family members, and other factors adding to the list of previously described factors that are involved in keratinocyte-specific gene expression. These studies highlight a cooperative interaction of the two HSs domains and strengthen the importance of combinatorial play of transcription factors that govern keratinocyte-specific gene regulation.

Customizing for clients: developing a library liaison program from need to plan*

Building on the experiences of librarian representatives to curriculum committees in the colleges of dentistry, medicine, and nursing, the Health Science Center Libraries (HSCL) Strategic Plan recommended the formation of a Library Liaison Work Group to create a formal Library Liaison Program to serve the six Health Science Center (HSC) colleges and several affiliated centers and institutes. The work group's charge was to define the purpose and scope of the program, identify models of best practice, and recommend activities for liaisons. The work group gathered background information, performed an environmental scan, and developed a philosophy statement, a program of liaison activities focusing on seven |primary areas, and a forum for liaison communication. Hallmarks of the plan included intensive subject specialization (beyond collection development), extensive communication with users, and personal information services. Specialization was expected to promote competence, communication, confidence, comfort, and customization. Development of the program required close coordination with other strategic plan implementation teams, including teams for collection development, education, and marketing. This paper discusses the HSCL's planning process and the resulting Library Liaison Program. Although focusing on an academic health center, the planning process and liaison model may be applied to any library serving diverse, subject-specific user populations.

In vivo restoration of laminin 5 β3 expression and function in junctional epidermolysis bullosa

The blistering disorder, lethal junctional epidermolysis bullosa (JEB), can result from mutations in the LAMB3 gene, which encodes laminin 5 β3 (β3). Appropriate expression of LAMβ3 in JEB skin tissue could potentially ameliorate the symptoms of the underlying disease. To explore the utility of this therapeutic approach, primary keratinocytes from six unrelated JEB patients were transduced with a retroviral vector encoding β3 and used to regenerate human skin on severe combined immunodeficient (SCID) mice. Tissue regenerated from β3-transduced JEB keratinocytes produced phenotypically normal skin characterized by sustained β3 expression and the formation of hemidesmosomes. Additionally, β3 gene transfer corrected the distribution of a number of important basement membrane zone proteins including BPAG2, integrins β4/β1, and laminins α3/γ2. Skin produced from β3-negative (β3[−]) JEB cells mimicked the hallmarks of the disease state and did not exhibit any of the aforementioned traits. Therefore, by effecting therapeutic gene transfer to β3-deficient primary keratinocytes, it is possible to produce healthy, normal skin tissue in vivo. These data support the utility of gene therapy for JEB and highlight the potential for gene delivery in the treatment of human genetic skin disease.

Genome-wide gene expression profiles of the developing mouse hippocampus

We have analyzed the developmental molecular programs of the mouse hippocampus, a cortical structure critical for learning and memory, by means of large-scale DNA microarray techniques. Of 11,000 genes and expressed sequence tags examined, 1,926 showed dynamic changes during hippocampal development from embryonic day 16 to postnatal day 30. Gene-cluster analysis was used to group these genes into 16 distinct clusters with striking patterns that appear to correlate with major developmental hallmarks and cellular events. These include genes involved in neuronal proliferation, differentiation, and synapse formation. A complete list of the transcriptional changes has been compiled into a comprehensive gene profile database (http://BrainGenomics.Princeton.edu), which should prove valuable in advancing our understanding of the molecular and genetic programs underlying both the development and the functions of the mammalian brain.

Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.

Fumonisins and Alternaria alternata lycopersici toxins: sphinganine analog mycotoxins induce apoptosis in monkey kidney cells.

Fusarium moniliforme toxins (fumonisins) and Alternaria alternata lycopersici (AAL) toxins are members of a new class of sphinganine analog mycotoxins that occur widely in the food chain. These mycotoxins represent a serious threat to human and animal health, inducing both cell death and neoplastic events in mammals. The mechanisms by which this family of chemical congeners induce changes in cell homeostasis were investigated in African green monkey kidney cells (CV-1) by assessing the appearance of apoptosis, cell cycle regulation, and putative components of signal transduction pathways involved in apoptosis. Structurally, these mycotoxins resemble the sphingoid bases, sphingosine and sphinganine, that are reported to play critical roles in cell communication and signal transduction. The addition of fumonisin B1 or AAL toxin, TA, to CV-1 cells induced the stereotypical hallmarks of apoptosis, including the formation of DNA ladders, compaction of nuclear DNA, and the subsequent appearance of apoptotic bodies. Neither mycotoxin induced cell death, DNA ladders, or apoptotic bodies in CV-1 cells expressing simian virus 40 large T antigen (COS-7) at toxin concentrations that readily killed CV-1 cells. Fumonisin B1 induced cell cycle arrest in the G1 phase in CV-1 cells but not in COS-7 cells. AAL toxin TA did not arrest cell cycle progression in either cell line. The induction of apoptosis combined with the widespread presence of these compounds in food crops and animal feed identifies a previously unrecognized health risk to humans and livestock. These molecules also represent a new class of natural toxicants that can be used as model compounds to further characterize the molecular and biochemical pathways leading to apoptosis.