A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

A Monte Carlo investigation of homogeneity tests of the odds ratio under various sample size configurations

Epidemiologic data for case-control studies are often summarized into K 2 x 2 tables. Given a fixed number of cases and controls, the degree of sparseness in the data depends on the number of strata, K. The effect of increasing stratification on size and power of seven tests of homogeneity of the odds ratio is studied using Monte Carlo methods. In all the designs considered here, the numbers of cases and controls per stratum are the same. Considering both size and power in non-sparse-data settings, we recommend the Breslow-Day statistic (1980, Statistical Methods in Cancer Research, 1. The Analysis of Case-Control Studies, p. 142; Lyon: International Agency for Research on Cancer) for general use. In sparse-data settings the T4 statistic of Liang and Self (1985, Biometrika 72, 353-358) performs the best when all tables, regardless of sample size, have odds ratios generated from the same distribution. In sparse-data settings characterized by a large table with an odds ratio of 1 and many small tables with odds ratios greater than 1, the T5 statistic of Liang and Self (1985) performs the best. One of the most important results of this study is the generally low power for all homogeneity tests especially when the data are sparse.     

Mathematical descriptions of hybridoma culture kinetics: II. The relationship between thiol chemistry and the degradation of serum activity

The growth rate of the hybridoma cell line ATCC-CRL-1606 in low serum medium declines rapidly with time after inoculation. To characterize this phenomenon, the stability of the growth-promoting activity of serum was investigated. The activity of serum was found to de grade with time, and was stabilized by alterations in the medium formulation that acted to lower the oxidation/reduction potential. This included both the addition of thiols and the elimination of disulfides from the medium. Additionally, cysteine and other thiols were shown to stimulate growth in low serum, low cell density cultures, suggesting that thiols may be rate-limiting in low serum medium. Stimulation of growth by thiol addition was less significant at high cell levels, implying that the cells themselves may be acting to reduce their environment. A hypothesis is presented based on these results which suggests that the actual rate-limiting moieties in low serum cultures may be dithiols.     

Classification of implants with reference to safety considerations

Implants are listed both in the German Drug Law (Arzneimittelgesetz) and in the Regulations of the Safety of Medical Technical Equipment (Medizinger?teverordnung). In recent time, various guidelines concerning implants have been under discussion in Europe. It is surprising to note that more than one set of guidelines has been thought to be necessary. The present article discusses possible definitions of some of the terms employed in connection with implants, and attempts to identify the differences and common features of implants under aspects of safety, thus providing a broader base for public discussions. An analysis of the implants presently available has shown that it is useful to establish a hierarchy of possible hazards that takes into account the danger to the patient under worst-case conditions. A survey has demonstrated that in the worst case, most implants are associated with only an impairment or inconvenience for the patient, but no threat to life. In contrast, a minority of implants are of such a nature that in the case of severe failure, a life-threatening situation can develop for the patient. Such a hazard is not dependent upon whether the implant has a driving energy source or not. These findings indicate that within the framework of safery considerations, more attention need to be paid to "passive" implants than has been the case so far.     

Oxacillin-hydrolysing beta-lactamases. A comparative analysis at nucleotide and amino acid sequence levels

We have extended the sequence of the OXA-2 beta-lactamase which together with S1 mapping has enabled us to identify the promoter site for this gene. This lies in a region that is found upstream from a variety of resistance genes on different plasmids; each gene appears to have been inserted at the same specific site and to be expressed from the same promoter. The ancestral plasmid thus appears to function as a natural expression vector. The sequence of the recombination site at the 5' end of the OXA-2 gene shows a marked similarity with the attP sequence of lambda. DNA-probe analysis confirmed that the OXA-2 and OXA-3 beta-lactamases are related, and indicated no similarity with other beta-lactamase genes. However, a comparison of amino acid sequences demonstrates that the OXA-2, OXA-1 and PSE-2 beta-lactamases show some similarities to the typical class A enzymes, especially in the central helical domain of the latter, which is largely responsible for forming the active site of the enzyme. The three oxacillinases also show marked amino acid sequence similarity with the product of a regulatory gene, blaR1, required for beta-lactamase induction in Bacillus licheniformis.     

Mechanism and binding specificity of beta-glucosidase-catalyzed hydrolysis of cellobiose analogues studied by competition enzyme kinetics monitored by 1H-NMR spectroscopy

The application of high-resolution 1H-NMR spectroscopy to monitor substrate and product time dependencies in progress curve enzyme kinetics is described with beta-glucosidase-catalyzed hydrolyses of cellobiose analogues as examples. It is demonstrated that inhibition patterns, relative binding specificities and catalytic rates can be inferred from competition experiments with two or more substrates. It could be concluded from competition experiments that substrates which form less stable enzyme-substrate complexes than methyl beta-cellobioside are hydrolyzed faster than this reference substrate when they are the sole substrate, due to a lower activation energy in the catalytic step, but that they are hydrolyzed slower than the reference compound in direct competition, due to the formation of the less stable enzyme-substrate complex in the binding step.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

Interactive analysis of phylogeny and character evolution using the computer program MacClade

Computer programs for phylogenetic analysis have been important tools in systematics and evolutionary biology, but most have been designed primarily for the reconstruction of phylogenetic trees and not the interpretation of patterns of character evolution. Described here is the computer program MacClade, designed for interactive analysis of character evolution and phylogeny. For a given tree and a matrix of character data, MacClade displays its reconstruction of character evolution by shading the branches of the tree to indicate ancestral states. Trees can be manipulated for instance by picking up and moving branches. Assumptions underlying the reconstruction of character evolution can be varied extensively. With these manipulations and MacClade's graphical feedback, one can explore the relationships among phylogenetic trees, character data, assumptions and interpretations of character evolution. MacClade has extensive facilities for editing data, displaying various summaries of character evolution in charts and diagrams, and printing.     

Functional differentiation of long bones in lorises

The external dimensions of the limb bones and the geometry of their midshaft cross-sections were determined for Loris tardigradus and Nycticebus coucang. Relative cortical thickness, cortical area, and second moment of area were calculated and contrasted with locomotor stresses. The difference in shape-related strength of the bones between the smaller- and the larger-bodied species is more pronounced than can be expected from stresses acting during normal locomotion. The Nycticebus skeleton has a much higher safety margin overall and seems to be dimensioned for infrequent but critical stresses of high magnitude. Lorisine gaits in general are characterized by low ground reaction forces, great mobility in all joints, and a nearly equal share in propulsion and weight-bearing by the fore- and hindlimb. Accordingly, the long bones of lorises (especially those of L. tardigradus) tend to be less rigid than those of other mammalian species (including other primates), they lack a preferential plane of higher bending strength, and femur and humerus do not differ markedly in their capacity to withstand mechanical stresses. External dimensions of the humerus and femur of the two African lorisine species parallel and corroborate these results. Some more general implications for the relationships between bone shape and locomotor stresses are also discussed.     

Random sample planning in morphometry

This report tries to explain principles and presuppositions of planning sample sizes. The aim is estimation of the optimal, that means minimally necessary sample size, justifying an investigation ethically and economically. The problems are complicated because of the necessity of nested sampling or samples within samples. We describe the most important presuppositions using an example of mean value estimation. These are: establishing of demands for exactness, of error probability, and knowledge of variance of the parameters. It is explained in detail by means of binomially distributed variables as we find them in the point counting method.