Dynamic regulation of polysialylated neural cell adhesion molecule in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) prominently expresses polysialic acid (PSA), a carbohydrate polymer that is attached to neural cell adhesion molecule (NCAM) and promotes changes in cell interactions. Previous studies have shown that expression of PSA is important for circadian rhythm stability under constant darkness, and for photic entrainment of the SCN circadian clock. In the present study, immunoblot analyses of the Syrian hamster SCN revealed marked diurnal fluctuations in PSA under a 24-h light/dark cycle.

Intrinsic role of polysialylated neural cell adhesion molecule in photic phase resetting of the Mammalian circadian clock.

The suprachiasmatic nuclei (SCN), the location of the mammalian circadian clock, are one of the few adult brain regions that express the highly polysialylated form of neural cell adhesion molecule (PSA-NCAM). A role for the polysialic acid (PSA) component of PSA-NCAM, which is known to promote tissue plasticity, has been reported for photic entrainment of circadian rhythmicity in vivo. The in vivo results, however, do not discriminate between PSA acting upstream or downstream of the glutamatergic synapses that convey photic information to the SCN.

Carbohydrate vaccines in cancer: immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man.

The complex carbohydrate molecule globo H hexasaccharide has been synthesized, conjugated to keyhole limpet hemocyanin, and administered with the immunologic adjuvant QS-21 as a vaccine for patients with prostate cancer who have relapsed after primary therapies such as radiation or surgery. Globo H is one of several candidate antigens present on prostate cancer cells that can serve as targets for immune recognition and treatment strategies. The vaccine, given as five subcutaneous vaccinations over 26 weeks, has been shown to be safe and capable of inducing specific high-titer IgM antibodies against globo H.

Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA.

Retrovirus-mediated gene transfer was used to restore expression to MHC class II-negative patient cells from complementation group A(II) of MHC class II immunodeficiency or bare lymphocyte syndrome (BLS). The cells of these patients do not transcribe MHC class II genes due to a defect in the trans-acting factor, CIITA. We constructed a vector, pGAG/Ii-CIITA, with the MHC class II-associated invariant chain promoter driving CIITA expression.

Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells.

Overexpression of sequences corresponding to the major Rev-binding site in the Rev response element of human immunodeficiency virus type 1 (HIV-1) (RRE decoys) was used to render cells resistant to HIV-1 replication. This was accomplished by the use of a chimeric tRNA-RRE transcription unit in a double-copy murine retroviral vector to express high levels of HIV-1 RRE-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited more than 90% in cells expressing chimeric tRNA-RRE transcripts, as determined by in situ immunofluorescence analysis and a p24 antigen ELISA test.

Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication.

An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals.

Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat.

Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication.

Overexpression of TAR-containing sequences (TAR decoys) was used to render cells resistant to HIV replication. A chimeric tRNA(meti)-TAR transcription unit contained in a double copy murine retroviral vector was used to express high levels of HIV-1 TAR-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited over 99% in cells expressing chimeric tRNA-TAR transcripts, but an amphotropic murine retrovirus replicated normally in these cells.

Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.

Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR.

Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes.

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection.

Effects of benzo[a]pyrene diol epoxide adducts on DNA synthesis in mammalian cells.

The replication of DNA containing anti-benzo[a]pyrene diol epoxide (BPDE) adducts was studied in mammalian cells by first treating SV40 virus with BPDE in vitro, then infecting cells with virus containing a known number of adducts in the DNA. Viral transcription products necessary for replication were supplied by co-infection with an untreated virus containing a deletion as a DNA marker. Thus, only replicative effects of BPDE adducts were manifested.

Growth and purification of SV40 virus for biochemical studies.

A detailed growth and purification scheme suitable for producing relatively large quantities of fully active, pure SV40 is presented together with data on recovery and purity at each step of the procedure. The scheme was designed to prevent the initial binding of virus to cell components as well as contamination of the extracted virus by cellular DNA.

DNA swivel enzyme activity in a nuclear membrane fraction.

DNA swivel (nicking-rejoining) enzyme activity has been studied in various cell fractions of a human lymphoid cell line. Swivel activity is found only in chromatin and in a nuclear membrane fraction containing DNA and possessing endogenous DNA synthesizing activity. Twenty percent of the total swivel activity and less than one percent of the total DNA are in the membrane fraction.

Variation in DNA swivel enzyme activity during the mammalian cell cycle.

Synchronized cells of a normal human lymphocytic cell line contain little swiven enzyme activity in G0 and G1 and high activity in Sphase. The level of activity in different growth phase appears to be related to the fraction of the population engaged in DNA replication. No endogenous inhibitor or activator of swiven activity could be demonstrated.

The negative control mechanism for E. coli DNA replication.

Evidence is presented to show that the initiation of DNA replication in E. coli 555-7 requires synthesis of a protein whose production is correlated with total protein synthesis. Once replication is initiated, however, reinitiation will occur if all further protein synthesis is prevented; a small amount of protein synthesis is sufficient to prevent this unregulated reinitiation.

Isolation and characterization of carotenoid pigments of Micrococcus roseus.

In addition to canthaxanthin, seven pigment fractions were isolated from Micrococcus roseus. They were purified by solvent partitioning and by column and thin-layer chromatography. Visible absorption spectra, chromatographic behavior, and partition coefficients of the pigments and derivatives prepared from the pigments were used in characterizing them.