Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

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.     

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.     

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.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Quantitative high-pressure liquid chromatographic analysis of methylated purines in DNA of rats treated with chemical carcinogens

A rapid, sensitive method for the quantitative measurement of certain major and modified purines in DNA of carcinogen-treated animals is presented. DNA hydrolysates are analyzed by high-pressure liquid chromatography combined with fluorescence detection and electronic integration of peaks. Limits of detection are approximately 7 ng for 7-methylguanine and 150 pg for O⁶-methylguanine. Between 100 and 250 μg target organ DNA from animals treated with several carcinogens was shown to contain readily detectable amounts of these methylated bases. The method provides results comparable to those obtained with conventional methods using radioactively labeled carcinogens.