No abstract

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n ‫؍‬ 72), C. dubliniensis (n ‫؍‬ 58), C. glabrata (n ‫؍‬ 5), C. krusei (n ‫؍‬ 2), C. parapsilosis (n ‫؍‬ 4), and C. tropicalis (n ‫؍‬ 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.

A comparison of seven human DR and DC class II histocompatibility antigen beta‐chain amino acid sequences indicates that the allelic variation is of comparable magnitude within the DR and DC beta‐chain genes. Silent and replacement nucleotide substitutions in six DR and DC beta‐chain sequences, as well as in seven murine class II sequences (three I‐A beta and four I‐A alpha alleles) were analyzed. The results suggest that the mutation rates are of a comparable magnitude in the nucleotide sequences encoding the first and second external domains of the class II molecules. Nevertheless, the allelic amino acid replacements are predominantly located in the first domains. We conclude that a conservative selective pressure acts on the second domains, whereas in many positions in the first domains replacement substitutions are selectively neutral or maybe even favoured. Thus, the difference between the first and second domains as regards the number of amino acid replacements is mainly due to selection.

Establishing a general and effective method for regulating gene expression in mammalian systems is important for many aspects of biological and biomedical research. Herein we report the antisense activities of a cell-permeable, guanidine-based peptide nucleic acid (PNA) called GPNA. We show that a GPNA oligomer designed to bind to the transcriptional start-site of human E-cadherin gene induces potent and sequence-specific antisense effects and is less toxic to the cells than the corresponding PNA-polyarginine conjugate. GPNA confers its silencing effect by blocking protein translation. The findings reported in this study provide a molecular framework for designing the next generation cell-permeable nucleic acid mimics for regulating gene expression in live cells and intact organisms.

We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences which interrupt the two coding sequences in identical positions. The 5' and 3' flanking sequences in both genes contain conserved sequences similar to those found in corresponding positions in other eukaryotic genes. Thus, the general DNA sequence organization of these plant genes is similar to that of other eukaryotic genes.