Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity

Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies. We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection. The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin. The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology. Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins. This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy. We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.     

The Potential Importance of Mine Sites for Biodiversity

Abstract  Abandoned mine sites are typically viewed as environmental problems due to their negative impacts on local ecosystems. This paper presents an alternative viewpoint providing evidence of the potential importance of mine sites for supporting rare and threatened species from many of the major taxonomic orders. The potential importance of these species in remediation of polluted environments is also highlighted.     

Effects of intermittent cycle exercise on intramyocellular lipid use and recovery

The purpose of this investigation was to compare intramyocellular lipid (IMCL) changes in skeletal muscle in nine moderately trained subjects after 45 min of interval cycling and through 1 h of recovery. The exercise session was continous with alternating cycling intensity achieving 50 (3 min) and 110% (2 min) of ventilatory threshold. Spectra from the vastus lateralis were acquired before, immediately after, and 60 min following exercise using a 1.5 T Signa whole-body magnet (point-resolved spectroscopy sequence, echo time 60 ms, transverse relaxation time 2000 ms, 128 acquisitions, and 20 mm³ voxel). Immediately following exercise, IMCI concentration decreased 38% compared to pre-exercise levels (P<0.05). Fitness level and baseline IMCL were not correlated with changes in IMCL following exercise (P>0.05). In the 60-min recovery, IMCL was reduced 30% compared to baseline (P<0.05) and did not recover. In contrast, a nonexercising control group showed no change in IMCL. Our results suggest that IMCL decreased significantly following 45 min of interval cycling, with little recovery in the hour following.     

Sex differences in human motor activity level.

Examined sex differences in activity level (AL) by quantitatively integrating results from 90 citations encompassing 127 independent sex-difference studies. A variety of developmental, situational, measurement, and investigator factors thought to be related to the AL sex-difference magnitude was assessed. Sample sizes ranged from 7 to 25,000; chronological ages ranged from 2 mo before birth to 30 yrs. A scale-independent index of effect size (d) was used to summarize the results of a between-sexes comparison, and ds across investigations were aggregated with measures of AL. Findings indicate that males were generally more active than females, although the magnitude of the difference was associated with other features of the research investigation, such as participant age and situational characteristics. This AL result was judged to be a large effect within the context of established behavioral sex differences. Data are consistent with the view that social influences enlarge AL sex differences. A list of citations used in the study is appended. (32 ref) (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.     

Glyoxal as a possible curing agent for paints based on sodium silicate

The effects of adding glyoxal to sodium silicate solutions have been investigated and compared with those of adding glycollic acid or hydrochloric acid: gel-time and the proportions of small silicate units in the solutions were both studied. It is concluded that the effect of adding glycollic acid is the same as that of adding HCI and that the effect of adding glyoxal is wholly accounted for by its conversion to glycollic acid through a Cannizzaro reaction.