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July 2002

Regenerative Medicine

Cytokines and neurotrophic factors fail to affect Nogo-A mRNA expression in differentiated human neurones: implications for inflammation-related axonal regeneration in the central nervous system.

Nogo is a novel myelin-associated inhibitor of neurite outgrowth which regulates stable neuronal connections during axonal regeneration following injury in the adult mammalian central nervous system (CNS). Because cytokines and neurotrophic factors play a key role in inflammation-related axonal regeneration, we investigated: (i) the constitutive expression of Nogo and the Nogo receptor (NgR) mRNA in human neural cell lines; (ii) Nogo and NgR mRNA levels in the NTera2 human teratocarcinoma cell line during retinoic acid (RA)-induced neuronal differentiation; and (iii) their regulation in NTera2-derived differentiated neurones (NTera2-N) after exposure to a battery of cytokines and growth factors potentially produced by activated glial cells at post-traumatic inflammatory lesions in the CNS. By reverse transcriptase-polymerase chain reaction analysis, the constitutive expression of Nogo-A, the longest isoform of three distinct Nogo transcripts and NgR mRNA was identified in a wide variety of human neural and non-neural cell lines. By Northern blot analysis, the levels of Nogo-A mRNA were elevated markedly in NTera2 cells following RA-induced neuronal differentiation, accompanied by an increased expression of the neurite growth-associated protein GAP-43 mRNA. In contrast, Nogo-A, Nogo-B, NgR and GAP-43 mRNA levels were unaltered in NTera2-N cells by exposure to basic fibroblast growth factor, brain-derived neurotrophic factor, glia-derived neurotrophic factor, tumour necrosis factor-alpha, interleukin-1beta, dibutyryl cyclic AMP or phorbol 12-myristate 13-acetate. These results indicate that both Nogo-A and NgR mRNA are coexpressed in various human cell types, including differentiated neurones, where their expression is unaffected by exposure to a panel of cytokines and neurotrophic factors which might be involved in inflammation-related axonal regeneration in the CNS.

Neuropathol Appl Neurobiol 2002 Apr;28(2):95-106

Messenger-RNA-binding proteins and the messages they carry.

From sites of transcription in the nucleus to the outreaches of the cytoplasm, messenger RNAs are associated with RNA-binding proteins. These proteins influence pre-mRNA processing as well as the transport, localization, translation and stability of mRNAs. Recent discoveries have shown that one group of these proteins marks exon-exon junctions and has a role in mRNA export. These proteins communicate crucial information to the translation machinery for the surveillance of nonsense mutations and for mRNA localization and translation.

Nat Rev Mol Cell Biol 2002 Mar;3(3):195-205

cDNA cloning confirms the polyadenylation of RNA decay intermediates in Streptomyces coelicolor.

In Escherichia coli the poly(A) tails of messenger and rRNAs are a major determinant of RNA stability. These tails are formed primarily by poly(A) polymerase I (PAP I) in wild-type strains or by polynucleotide phosphorylase (PNPase) in PAP I-deficient strains. In Streptomyces coelicolor it has been shown that mycelial RNAs display biochemical characteristics consistent with the presence of poly(A) tails. To confirm the occurrence of polyadenylation, rRNA and mRNA transcripts from S. coelicolor were isolated by oligo(dT)-dependent RT-PCR followed by cDNA cloning. One of the clones obtained was polyadenylated at a site corresponding to the mature 3’ terminus of 16S rRNA, while two 23S rRNA cDNA clones were polyadenylated at precursor processing sites. Other clones identified polyadenylation sites internal to the coding regions of both 16S and 23S rRNAs, and redD and actII-orf4 mRNAs. While most rRNA cDNA clones displayed adenosine homopolymer tails, the poly(A) tails of three rRNAs and all the redD and actII-orf4 clones consisted of a variety of heteropolymers. These results suggest that the enzyme primarily responsible for polyadenylation in S. coelicolor is PNPase rather than a PAP I homologue.

Microbiology 2002 May;148(Pt 5):1421-5

Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.

Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesized against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3’-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3’ mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

Nucleic Acids Res 2002 Apr 15;30(8):1757-66

A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli.

A small RNA, RyhB, was found as part of a genomewide search for novel small RNAs in Escherichia coli. The RyhB 90-nt RNA down-regulates a set of iron-storage and iron-using proteins when iron is limiting; it is itself negatively regulated by the ferric uptake repressor protein, Fur (Ferric uptake regulator). RyhB RNA levels are inversely correlated with mRNA levels for the sdhCDAB operon, encoding succinate dehydrogenase, as well as five other genes previously shown to be positively regulated by Fur by an unknown mechanism. These include two other genes encoding enzymes in the tricarboxylic acid cycle, acnA and fumA, two ferritin genes, ftnA and bfr, and a gene for superoxide dismutase, sodB. Fur positive regulation of all these genes is fully reversed in an RyhB mutant. Our results explain the previously observed inability of fur mutants to grow on succinate. RyhB requires the RNA-binding protein, Hfq, for activity. Sequences within RyhB are complementary to regions within each of the target genes, suggesting that RyhB acts as an antisense RNA. In sdhCDAB, the complementary region is at the end of the first gene of the sdhCDAB operon; full-length sdhCDAB message disappears and a truncated message, equivalent in size to the region upstream of the complementarity, is detected when RyhB is expressed. RyhB provides a mechanism for the cell to down-regulate iron-storage proteins and nonessential iron-containing proteins when iron is limiting, thus modulating intracellular iron usage to supplement mechanisms for iron uptake directly regulated by Fur.

Proc Natl Acad Sci U S A 2002 Apr 2;99(7):4620-5

Lymphocyte autoantibodies and alloantibodies in HIV-positive haemophilia patients.

Immune parameters were studied in 86 haemophilia patients (six with AIDS) and 87 healthy controls. We found lymphocytotoxic alloantibodies in HIV-positive (HIV+) sera, which reacted preferentially with B lymphocytes but also with T lymphocytes, and which reacted more frequently at 4 degrees C than at 37 degrees C. The antibodies were not directed against HIV-induced structures on T lymphocytes and they were reactive with both CD4+ and CD8+ lymphocytes. In addition to cytotoxic alloantibodies, cytotoxic autoantibodies were detected, which coated patient lymphocytes in vivo. Increased proportions of in vivo-antibody-coated-cells were found in 37 of 86 haemophilia patients. Antibody binding was labile so that the immunoglobulins were partially removed from the lymphocyte surface by washing. The autoreactive antibodies were of IgG and IgM type, fixed complement as demonstrated by increased anti-C3d+ cells in the patients’ blood, and reacted with CD4+ as well as CD8+ lymphocytes. There was a statistically significant correlation of increased Ig+ cells with HIV infection, decreased CD4/CD8 ratios, increased serum neopterin levels, and abnormal in-vitro responses to pooled allogeneic stimulator cells or CD3 monoclonal antibody. Patients with increased Ig+ cells were lymphopenic, had decreased absolute counts of CD4+, CD25+, CD21+ and OKM5+ cells, and higher percentages of CD8+ and OKIa1+ cells in their blood than patients with normal levels of Ig+ cells. Our data suggest a role of autoreactive anti-lymphocyte antibodies in the pathogenesis of acquired immunodeficiency.

Clin Exp Immunol 1989 Feb;75(2):178-83

Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice.

We present genome-wide microarray expression analysis of 11,000 genes in an aging potentially mitotic tissue, the liver. This organ has a major impact on health and homeostasis during aging. The effects of life- and health-span-extending caloric restriction (CR) on gene expression among young and old mice and between long-term CR (LT-CR) and short-term CR (ST-CR) were examined. This experimental design allowed us to accurately distinguish the effects of aging from those of CR on gene expression. Aging was accompanied by changes in gene expression associated with increased inflammation, cellular stress, and fibrosis, and reduced capacity for apoptosis, xenobiotic metabolism, normal cell-cycling, and DNA replication. LT-CR and just 4 weeks of ST-CR reversed the majority of these changes. LT-CR produced in young mice a pattern of gene expression that is a subset of the changes found in old LT-CR mice. It is possible that the early changes in gene expression, which extend into old age, are key to the life- and health-span-extending effects of CR. Further, ST-CR substantially shifted the "normo-aging" genomic profile of old control mice toward the "slow-aging" profile associated with LT-CR. Therefore, many of the genomic effects of CR are established rapidly. Thus, expression profiling should prove useful in quickly identifying CR-mimetic drugs and treatments.

Proc Natl Acad Sci U S A 2001 Sep 11;98(19):10630-5