Construction and characterization of Escherichia coli disruptants defective in the yaeM gene.

Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli.     

In vitro assessment of the toxicity of metal compounds

A review of in vitro mutagenesis assessment of metal compounds in mammalian and nonmammalian test systems has been compiled. Prokaryotic assays are ineffective or inconsistent in their detection of most metals as mutagens, with the notable exception of hexavalent chromium. Mammalian assay systems appear to be similarly inappropriate for the screening of metal compounds based upon the limited number of studies that have employed those compounds having known carcinogenic activity. Although of limited value as screening tests for the detection of potentially carcinogenic metal compounds, the well-characterized in vitro mutagenesis systems may prove to be of significant value as a means to elucidate mechanisms of metal genotoxicity.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

Cyanobacteria passage through drinking water filters during perturbation episodes as a function of cell morphology, coagulant and initial filter loading rate

Eight pilot-scale in-line filtration trials were performed to evaluate the passage of cyanobacterial cells through drinking water filters after sudden increases in hydraulic loading rates. Trials were performed at 30 °C using two coagulant combinations (aluminum sulfate and cationic polymer or ferric chloride and cationic polymer), two initial filter loading rates (7 or 10 m/h) and two species of morphologically different cyanobacteria (Microcystis aeruginosa or Anabaena flos aquae). The filter was perturbed by instantaneously increasing the hydraulic loading rate by 50%. Filter influent and effluent water qualities were characterized by measuring turbidity, particles and chlorophyll a. The observed post-perturbation filter effluent chlorophyll a peaks were 1.6–48 times greater than the pre-perturbation averages. Chlorophyll a peaks were larger for M. aeruginosa than for A. flos aquae. Chlorophyll a peaks were also larger for the higher (10 m/h) than for the lower (7 m/h) initial filter loading rate. The post-perturbation effluent turbidity peaks were 1.4–7.2 times greater than the pre-perturbation averages. The post-perturbation effluent particle peaks were 6.5–25 times greater than the pre-perturbation averages. These results indicate that particles were a more sensitive indicator of cyanobacterial passage than turbidity.     

Warm temperatures lead to early onset of incubation, shorter incubation periods and greater hatching asynchrony in tree swallows Tachycineta bicolor at the extremes of their range

The onset of incubation varies in birds, with many species beginning incubation prior to clutch completion. Here we examine whether early onset is more likely to occur during high temperatures, a critical prediction of the egg-viability hypothesis, which suggest that birds begin incubation prior to clutch completion in order to maintain egg-viability. We examined onset of incubation in tree swallows Tachycineta bicolor at two locations at the extremes of their breeding range, Alaska and Tennessee. A majority of individuals (68%) began incubation prior to clutch completion. While females in Tennessee were more likely to begin incubation early, there was no difference between sites when differences in temperatures inside nestboxes were controlled in analyses. Rather, early onset of incubation was predicted by the proportion of daily temperatures above physiological zero during laying, a critical prediction of the egg viability hypothesis. Both warm weather and early onset led to shorter incubation periods and increased levels of hatching asynchrony. We found no effect of timing of nesting, female body condition index or clutch size on the probability of beginning incubation prior to clutch completion. Our results are consistent with the egg viability hypothesis, not consistent with a threshold clutch size rule, and do not support the hurry-up hypothesis, that individuals breeding later in the season would begin incubation early to reduce the time spent nesting. Overall, our results suggest that broad scale geographic differences in incubation behaviour may be explained by individual-level responses to environmental conditions.