Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity

We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.     

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg)

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

T cell tolerance to self antigens in New Zealand hybrid mice with lupus-like disease

We determined if self-reactive T cells are able to escape thymic tolerance in autoimmune New Zealand mice. T cells utilizing V beta 17a and V beta 11 encoded receptors have been shown to be clonally eliminated in nonautoimmune mice expressing I-E because of their potential self-reactivity. Similarly, V beta 8.1+ and V beta 6+ T cells are tolerized in the thymus of nonautoimmune mice that express Mls-1a. These T cell subsets were quantitated in the lymph nodes and spleens of (NZB x NZW)F1 and (NZB x SWR)F1 mice. In young mice from both autoimmune strains, deletion was similar to that observed in control animals matched for I-Ed and Mls-1a expression. Furthermore, older female autoimmune mice with elevated levels of IgG antinuclear antibodies and severe lupus-like renal disease did not demonstrate evidence of a global tolerance defect. We also found that the levels of residual V beta 17a+ cells in MHC-matched control F1 strains were further reduced by up to 80% in autoimmune (NZB x SWR)F1 mice. The greater in vivo elimination corresponded to an enhanced ability of NZB spleen cells, compared with other H-2d spleen cells, to stimulate V beta 17a+ hybridomas in vitro. The increased stimulation in culture could not be attributed to quantitative differences in I-E Ag expression. The results suggest that autoreactive T cells have been eliminated in these autoimmune mice by normal mechanisms of self-tolerance. Furthermore, the data demonstrate the existence of an NZB minor locus not present in other H-2d strains that influences T cell repertoire and enhances stimulation of T cells potentially reactive to self class II MHC Ag.     

The fate of the fusarium mycotoxin zearalenone in maize cell suspension cultures

The fusarium mycotoxin zearalenone was transformed in cell suspension cultures of Zea mays giving α- and β-zearalenol and the β-D-glu cos ides of zearalenone and α- and β-zearalenol. The structure of zearalenone-4-β-D-glucopyranoside was determined by liquid — chromatography-mass spectrometry and specific hydrolysis with β-glucosidase. α- and β-zearalenol and their glucosides were identified by co chromatography using tic and HPLC and glucosidase — treatment Up to 50% of the mycotoxin added was bound to a non extractable or “bound” residue fraction. After treating this residue by a sequential cell wall fractionation procedure, zearalenone was found to be bound mainly to starch, hemicellulose, and lignin fractions.     

A laboratory-based method to measure relative pesticide and spray oil efficacy against broad mite, Polyphagotarsonemus latus (Banks)(Acari: Tarsonemidae)

Six pesticides and two spray oils were tested against Polyphagotarsonemus latus. The chemicals were evaluated under laboratory conditions, requiring the development of a novel bioassay method, which is reported here. The pesticide toxicities fell into three distinct groups, namely abamectin, conventional pesticides and oils. The relative pesticide toxicities at the LC₅₀ level were abamectin 4.9×10^-8 g ai l^-1, endosulfan 1.1×10^-3 g ai l^-1, fenpyroximate 2.3×10^-3 g ai l^-1, pyridaben 4.1×10^-3 g ai l^-1, tebufenpyrad 4.4×10^-3 g ai l^-1, dicofol 4.5×10^-3 g ai l^-1, petroleum spray oil 3.4×10^-1 g ai l^-1 and canola oil 4.1×10^-1 g ai l^-1. The calculation of the LC_99.9 values allows for resistance monitoring in P. latus and the suggested discriminating concentrations are abamectin 1.0×10^-4 g ai l^-1; endosulfan, pyridaben and dicofol 1.0×10^-1 g ai l^-1 fenpyroximate and tebufenpyrad 5.0×10^-1 g ai l^-1.     

Gene transfer is a major factor in bacterial evolution

Lateral gene transfer in four strains of Salmonella enterica has been assessed using genomic subtraction. Strain LT2 (subspecies I serovar Typhimurium) chromosomal DNA was used as target and subtracted by three subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi (M229), and a subspecies V strain (M321). Data from probing random cosmids of LT2 DNA with preparations of the residual LT2 DNA after subtraction were used to estimate the amounts of LT2 DNA not able to hybridize to strains S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629, and 778-1,286 kb, respectively. Several lines of evidence indicate that most of this DNA is from genes not present in strain M321 and not from genes that have diverged in sequence. The amounts correlate with the divergence of the four strains as revealed by multilocus enzyme electrophoresis and sequence variation of housekeeping genes. Sequence of 39 of the fragments from the M321 subtracted residual LT2 DNA revealed only six inserts of known gene function with evidence of both gain and loss of genes during the development of S. enterica clones. Sixteen of the 39 segments have 45% or lower G+C content, below the species average, but over half are within the normal range for the species. We conclude that even within a species, clones may differ by up to 20% of chromosomal DNA, indicating a major role for lateral transfer, and that on the basis of G+C content, a significant proportion of the DNA is from distantly related species.      

Clofentezine and hexythiazox resistance in Tetranychus urticae Koch in Australia

Clofentezine resistance in T. urticae was first confirmed in Australia in 1987 after Queensland glasshouse roses had been exposed to 40 applications of clofentezine over a 10 month period. Clofentezine resistance in this strain was extremely high (>2.500x) and conferred high level cross-resistance to the chemically unrelated compound hexythiazox. Clofentezine resistance in T. urticae was detected in pome fruit orchards in the Goulburn Valley, Victoria in 1988 and caused field control failure after 5–6 sprays. Resistance was subsequently detected in Adelaide during 1988 and the Bathurst Orange region, NSW in 1989. Clofentezine and hexythiazox resistance appears particularly stable making it difficult to manage.This work contributes in part for the fulfilment of the requirements of the degree of PhD. at the University of Sydney.