Incidence ofCladosporium, alternaria and total fungal spores in the atmosphere of melbourne (Australia) over three years

A Burkard spore trap was used to monitor daily fungal spore counts in the atmosphere of Melbourne (Australia) between October 1991 and December 1994. Annual total spore counts varied widely (range 345 770 in 1994 to 1 106 037 in 1992), of which approximately half were identified asCladosporium sp. and only about 1%Alternaria sp. Highest daily total spore counts were recorded late-summer through to mid-winter, probably corresponding to senescence of annual grasses and leaf fall. Spore counts were negatively correlated with rainfall (P<0.05), significantly correlated with average temperature (P<0.001), and showed a highly significant linear relationship with cumulative temperature throughout the year.     

Lactococcal proteinase maturation protein PrtM is a lipoprotein.

The production of enzymatically active proteinase by lactococci requires the joint presence of a proteinase gene, prtP, and a gene encoding a maturation protein, prtM. A 32-kDa protein produced by Escherichia coli upon expression of the prtM gene under the direction of the T7 RNA polymerase promoter was purified and used to obtain PrtM-specific antibodies. With these antibodies, immunogold labeling of lactococcal cells revealed that PrtM was associated with the lactococcal cell envelope. Western blot (immunoblot) analysis of whole lactococcal cells and isolated membrane vesicles indicated that PrtM was a membrane-associated protein. Radiolabeling of Lactococcus lactis with [3H]palmitic acid showed that PrtM was a lipoprotein. Partial secretion of PrtM into the culture medium was observed after Cys-24, the target residue for lipid modification, was replaced by an Ala residue by means of site-directed mutagenesis. This mutation did not affect proteinase activity.     

Ossification of the human fetal ilium.

Ossification of the ilium is similar to that of a long bone. It possesses three cartilaginous epiphyses and one cartilaginous process. Moreover, it undergoes peculiar osteoclastic resorption, comparable with that of the cranium bones. Asymmetrical ossification of the ilium, haversian bone remodelling and apposition of chondroid tissue posterosuperiorly to the acetabulum most probably emphasize the importance of mechanical factors in the morphogenesis of the hip bone during fetal life.     

Phytanic acid alpha-oxidation: accumulation of 2-hydroxyphytanic acid and absence of 2-oxophytanic acid in plasma from patients with peroxisomal disorders.

A stable isotope dilution method was developed for the measurement of 2-hydroxyphytanic acid and 2-oxophytanic acid in plasma. In plasma from healthy individuals and from patients with Refsum's disease, 2-hydroxyphytanic acid was found at levels less than 0.2 mumol/l, whereas the acid accumulated in plasma from patients with rhizomelic chondrodysplasia punctata, generalized peroxisomal dysfunction, and a single peroxisomal beta-oxidation enzyme deficiency. In plasma from both healthy controls and patients with peroxisomal disorders, 2-oxophytanic acid was undetectable. Four different groups of diseases were characterized with a defective phytanic acid alpha-oxidation and/or pristanic acid beta-oxidation: 1) Refsum's disease, with a defect at phytanic acid alpha-hydroxylation; 2) rhizomelic chondrodysplasia punctata, with a defect at 2-hydroxyphytanic acid decarboxylation; 3) generalized peroxisomal disorders, with defects at 2-hydroxyphytanic acid decarboxylation and at pristanic acid beta-oxidation; 4) single peroxisomal beta-oxidation enzyme deficiencies, with a defect at pristanic acid beta-oxidation, resulting in an impaired phytanic acid alpha-oxidation by inhibition. The results indicate that 2-hydroxyphytanic acid decarboxylation and pristanic acid beta-oxidation take place in peroxisomes.     

The Pseudomonas oleovorans alkBAC operon encodes two structurally related rubredoxins and an aldehyde dehydrogenase

The Pseudomonas oleovorans alkBAC operon encodes seven proteins, of which at least three are involved in alkane hydroxylase (alkBA) and alkanol dehydrogenase (alkC) activities. We have determined the nucleotide sequence of the 2.5-kilobase pair alkA region and analyzed the role of its translation products in alkane oxidation. The alkA region contains three coding sequences, encoding two related rubredoxins (alkF and alkG) of 14- and 18-kDa molecular mass and a 52-kDa aldehyde dehydrogenase (alkH). Deletion analysis indicated that neither the 14-kDa alkF gene product (rubredoxin 1) nor the amino-terminal part of the 18-kDa alkG gene product (rubredoxin 2) is required for alkane hydroxylase activity in vivo. The product of the alkH cistron restores growth of a P. oleovorans aldehyde dehydrogenase mutant on aliphatic alcohols and aldehydes. Its amino acid sequence shows considerable homology to previously characterized aldehyde dehydrogenases from mammalian and fungal origin. The nucleotide composition of the alk genes (47% G + C) differs considerably from the G + C content of the P. oleovorans genome suggesting that the alk regulon may originate from an unrelated organism.     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

PDMP Blocks Brefeldin A–induced Retrograde Membrane Transport from Golgi to ER: Evidence for Involvement of Calcium Homeostasis and Dissociation from Sphingolipid Metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not “rescue” the Golgi compartment.Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C₆-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C₆-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C₆-ceramide addition or counteracted by exogenous C₆-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571.The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.     

Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.

OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).     

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.