Viability of lyophilized fungal cultures

Results of a viability test on 17–18 years old lyophilized cultures are reported.     

A novel adherence assay for Bordetella pertussis using tracheal organ cultures

Abstract In a novel adherence model using tracheal rings removed from Papio anubis , we have demonstrated a functional role for the fimbriae of Bordetella pertussis . When compared to wild-type strains, B. pertussis mutants specifically deficient in fimbriae adhered less well to the tracheal rings but better to Vero (Green monkey kidney) cells. In contrast, mutants deficient in filamentous haemagglutinin (FHA) production had reduced adherence to both Vero cells and the tracheal rings. These observations indicate that the fimbriae of B. pertussis , like those of many other bacterial pathogens, may play an important role in the initial stages of colonisation.     

Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres

The viability of lyophilized cultures of Lactobacillus bulgaricus in skim milk, during storage at different temperatures, relative humidities, and atmospheres was investigated. Survival was greatest at 11% relative humidity and at 5°C. Indirect and direct evidence is presented supporting the hypothesis that membrane damage occurs during storage. Experiments on the lipid composition of the cell membrane demonstrate that changes occur with time that are probably the result of oxidation. A study on the lipid composition of the cell membrane by gas chromatography showed that the unsaturated/saturated fatty acid index changes with time during storage.     

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules.     

A semi-automated protein assay for cell cultures

A semi-automated modification of the protein determination procedure of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) is described. The assay is well suited to the analysis of the protein of adherent cultured cells. The procedure is carried out in 96-well microtest plates on protein solutions of 50 microliter or less, and can detect less than 0.5 micrograms of protein (equivalent to about 10(3) cultured cells). Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min.     

Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay

Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme’s properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.     

Rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.     

Performing nucleic acid reactions using predispensed lyophilized reaction mixtures

A system is described in which manipulations of nucleic acids are performed in wells containing predispensed lyophilized reaction mixtures requiring addition of only nucleic acid. This allows increased reproducibility for single-step reactions (e.g., restrictions and ligations), as well as improved productivity for complex reactions (e.g., sequencing). Enzymes, co-factors, nucleotides and buffers can be dried and stored at room temperature without loss of essential function. When used for DNA sequencing, hundreds of templates a day can be sequenced with the potential to determine megabase amounts of sequence per week.     

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

The sequence of the major portion of aBacillus cycloheptanicus strain SCH^T 16S rRNA gene is reported. This sequence suggests thatB. cycloheptanicus is genetically quite distinct from traditionalBacillus strains (e.g.,B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.The EMBL accession number for the 16S rRNA sequence is X51928.     

Development of assay system for immunoglobulin production regulatory factors using whole cell cultures of mouse splenocytes

We tried to establish an assay system for screening and assessment of immunoregulatory factors using whole cell cultures of mouse splenocytes and found that splenic adhesive cells markedly increased immunogobulin (Ig) production of splenocytes. In the absence of adhesive cells, lipopolysaccharides, pokeweed mitogen, and phytohemagglutinin stimulated the production of IgA, IgG, and IgM in a class-dependent manner. Adhesive cells increased more markedly Ig production of splenocytes stimulated with these mitogens. When mouse splenocytes were cultured with milk proteins in the absence of adhesive cells, lactoferrin, beta-lactoglobulin, alpha-casein, and beta-casein stimulated IgA and IgG production. Adhesive cells increased IgA production of splenocytes stimulated with milk proteins, especially. These results suggest that the assay system is useful for assessment of Ig production-regulating factors.