Mechanism of increased microbial autofluorescence by heat treatment.

The total direct count (TDC) microbial enumeration method is rapid and suitable for analysing environmental samples containing numerous un-culturable micro-organisms. Conventional TDC methods require the addition of a fluorescent stain and are thus unsuitable for automatic monitoring. We unexpectedly found that heated micro-organisms emit strong autofluorescence. This study was conducted to determine how heating enhances the autofluorescence of bacteria and fungi and to evaluate whether the phenomenon could be exploited to develop a new TDC method. Bacterial autofluorescence was augmented by heating cells at 200°C. ELISA results indicated that levels of advanced glycation end products (AGEs) increased in heated microbes. Catechin, an inhibitor of the Maillard reaction, disrupted the intensification of autofluorescence. These results suggest that the enhanced autofluorescence is associated with the formation of AGEs and that the reaction could be utilized as alternative probe in TDC methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Autofluorescence of bacteria and fungi was prominently intensified by heat treatment at 200°C. This phenomenon was associated with advanced glycation end products formed in micro-organisms via the Maillard reaction. The fluorescence signal was strong enough to be utilized as an alternative probe for fluorescent dye in the total direct count method. This phenomenon could be incorporated in an automatic apparatus for microbial enumeration, as it does not require staining.

Antisense oligonucleotide to NOR-1, a novel orphan nuclear receptor, induces migration and neurite extension of cultured forebrain cells.

We previously identified a novel orphan nuclear receptor referred to as NOR-1 from rat forebrain cells. This study examined the role of NOR-1 in primary cultured forebrain cells by selectively inhibiting NOR-1 expression by addition of antisense oligonucleotide to the culture media. Treating cells with the antisense oligomer resulted in the following dramatic morphological changes: (i) cell migration, (ii) extension of processes, and (iii) formation of cellular aggregates. Immunocytochemistry for microtubule-associated protein 2 revealed that the processes were filled with neurites growing from neuronal cells. These findings suggest that NOR-1 may be involved in the molecular mechanisms regulating neural differentiation.

Molecular cloning of a novel thyroid/steroid receptor superfamily gene from cultured rat neuronal cells.

Brain neuronal cells in primary culture provide one system for the analysis of neuronal development, including apoptosis. By PCR amplification, with primers designed from conserved sequences among nuclear receptors, a novel member of the thyroid/steroid receptor superfamily was identified from forebrain neuronal cells undergoing apoptosis and designated as NOR-1 (neuron derived orphan receptor). A cDNA clone of NOR-1 encodes a 68kDa, 628 amino acid residue-containing protein, with an amino acid sequence highly homologous to the Nur77 family in its DNA-binding domain and moderately so in its putative ligand-binding domain. Using mobility shift assay, we showed that the NOR-1 protein binds to the B1a response-element which has been identified as the target sequence of the Nur77 family, suggesting that three members of the Nur77 family may transactivate common target gene(s) at different situations.