Microbial contamination of hydrogel contact lenses

Bacterial contamination of contact lenses (CLs) may contribute to CL-related corneal infection and inflammation. This study reports CL biota over time during daily and extended wear. Microbial contamination of a 58% water, ionic hydrogel CL and a 38% water, non-ionic hydrogel CL was evaluated in an Australian and an Indian population. Fifty wearers were repeatedly sampled over 18 months. Overnight CL use did not alter the frequency of positive cultures, nor the spectrum of organisms compared with daily CL wear. There were no differences in type and frequency of CL contamination between the CL types. Positive cultures were more frequently recovered from the Indian population compared with the Australian population. Streptococcus spp. and Propionibacterium spp. were more frequently isolated from the Australian population. Fungi and Bacillus spp. were more frequently isolated from the Indian population. Normal CL biota alone cannot explain the increased rate of infection and inflammation in extended wear.     

Identification and quantitation of alditol acetates of neutral and amino sugars from mucins by automated gas-liquid chromatography

An automated GLC method has been described that allows easy identification and quantitation on a single column of the alditol acetates of the commonly occurring neutral and amino sugars found in epithelial mucins on a new high-temperature stable column. Due to various losses that occur during the derivatization procedure, a constant, termed LF, must be entered into the calculation of the amount of each sugar present. When this factor is applied, the correlation between colorimetric and GLC results is very good.     

Stimulation of mouse migration inhibitory factor (MIF) production form MSV-immune lymphocytes by soluble tumor-associated antigen: requirement for histocompatible macrophages.

Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCL. We have previously demonstrated that this response is T cell-dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this "cooperative function" to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted for some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occuring during B-lymphocyte differentiation. The extent of the deleted region was determined using probes from the various IGLV subgroups and they each cover at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage V135 to the distal part of the IGLV gene region. Since the deletions are relatively small, the cell line will be valuable for mapping IGLV genes in the distal part of this region.     

Signature-tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity

Signature-tagged mutagenesis (STM) is a method that has been used to screen for genes required for in vivo survival of pathogenic bacteria, but has not been used to investigate a eukaryotic pathogen in an animal model of disease. We have adapted STM to identify genes required for in vivo growth of the opportunistic fungal pathogen Aspergillus fumigatus. Using a mouse model of invasive pulmonary aspergillosis, we have isolated several mutant strains with defects in their ability to replicate in vivo. One strain unable to cause lethal infection was further characterized and found to have an insertion into the promoter of a gene (pabaA) encoding para-aminobenzoic acid synthetase, an enzyme catalyzing a late step in the biosynthesis of folate. The complete inability of this strain, and other pabaA- strains constructed in this study by targeted gene deletion, to cause lethal infection in mice confirms the importance of the folate synthesis pathway for in vivo survival of this pathogen. The successful application of STM to A. fumigatus demonstrates that in vivo genetic analysis of eukaryotic pathogens is feasible and could result in the identification of potential targets, such as para-aminobenzoic acid synthetase, for novel antifungal therapies.     

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.