Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus.

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.

Patterns of temperature adaptation in proteins from Methanococcus and Bacillus.

It has long been known that amino acid substitutions in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are not all symmetrical; for example, more aligned sites have lysine in mesophiles and arginine in thermophiles than have the opposite pattern. This is generally taken to indicate that certain amino acids are favored over others by selection at different temperatures. Previous comparisons of protein sequences from mesophiles and thermophiles have used relatively small numbers of sequences from a diverse array of species, meaning that only the most common amino acid substitutions could be examined and any taxon-specific patterns would be obscured. Here, we compare a large number of proteins between mesophiles and thermophiles in the archaeal genus Methanococcus and the bacterial genus Bacillus. Each genus exhibits dramatically asymmetrical substitution patterns for many pairs of amino acids. There are several pairs of amino acids for which one amino acid is favored in thermophilic Bacillus and the other is favored in thermophilic Methanococcus; this appears to result from the higher G + C content of the DNA of thermophilic Bacillus, a complication not seen in Methanococcus.

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys.

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.

Improved tests for heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence.

The neutral theory of molecular evolution predicts that the ratio of polymorphisms to fixed differences should be fairly uniform across a region of DNA sequence. Significant heterogeneity in this ratio can indicate the effects of balancing selection, selective sweeps, mildly deleterious mutations, or background selection. Comparing an observed heterogeneity statistic with simulations of the heterogeneity resulting from random phylogenetic and sampling variation provides a test of the statistical significance of the observed pattern. When simulated data sets containing heterogeneity in the polymorphism-to-divergence ratio are examined, different statistics are most powerful for detecting different patterns of heterogeneity. The number of runs is most powerful for detecting patterns containing several peaks of polymorphism; the Kolmogorov-Smirnov statistic is most powerful for detecting patterns in which one end of the gene has high polymorphism and the other end has low polymorphism; and a newly developed statistic, the mean sliding G statistic, is most powerful for detecting patterns containing one or two peaks of polymorphism with reduced polymorphism on either side. Nine out of 27 genes from the Drosophila melanogaster subgroup exhibit heterogeneity that is significant under at least one of these three tests, with five of the nine remaining significant after a correction for multiple comparisons, suggesting that detectable evidence for the effects of some kind of selection is fairly common.

Synthesis of bisindolylmaleimides using a palladium catalyzed cross-coupling reaction.

Bisindolylmaleimides are known to be potent and selective PKC inhibitors. A new synthesis of this class of compound is reported. The key step is a Suzuki cross-coupling reaction using a readily available indolylmaleimide triflate intermediate.

3-trifluoromethylcarbacephems: synthesis of broad spectrum antibacterial compounds.

The enhanced stability of the carbacephem nucleus over the corresponding cephalosporin nucleus has allowed the synthesis of 7-arylglycyl-3-trifluoromethyl-carbacephems. These unique carbacephems possess broad spectrum activity and high stability to both plasmid and chromosomally mediated beta-lactamases.

The use of high-throughput synthesis and purification in the preparation of a directed library of adrenergic agents.

A library of potential agonists and antagonists for adrenergic receptors was prepared using high-throughput solution-phase parallel synthesis. Traditional solution-phase reductive amination reactions followed by rapid purification by ion exchange chromatography yielded products with near-analytical purity. An array of ketones and amines, arranged in an 8 x 12 matrix, were combined to form 96 individual compounds.

Lack of geographic variation in anonymous nuclear polymorphisms in the American oyster, Crassostrea virginica.

Comparing geographic variation of noncoding nuclear DNA polymorphisms, which presumably are neutral to natural selection, with geographic variation of allozymes is potentially a good way to detect the effects of selection on allozyme polymorphisms. A previous study of four anonymous nuclear markers in the American oyster, Crassostrea virginica, found dramatic differences in allele frequency between the Gulf of Mexico and the Atlantic Ocean. In contrast, 14 allozyme polymorphisms were fairly uniform in frequency between the two areas. This led to the conclusion that all of the allozyme polymorphisms were kept uniform in frequency by balancing selection. To test the robustness of this pattern, six additional anonymous nuclear DNA polymorphisms were surveyed in oysters from Panacea, Fla, and Charleston, S.C. on the Gulf and Atlantic coasts, respectively. Unlike the previously studied DNA markers, the six DNA polymorphisms examined here show geographic variation that is not significantly greater than that of allozymes. The reason for the discrepancy between the two sets of DNA polymorphisms is unclear.

(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta.

Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-membered macrocycles containing a N-N'-bridged bisindolylmaleimide moiety is described. A panel of eight cloned human PKC isozymes (alpha, beta I, beta II, gamma, delta, epsilon, sigma, eta) was used to identify the series and optimize the structure and associated activity relationship. The dimethylamine analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene++ +-1,3(2H)-dione, inhibits the PKC beta I (IC50 = 4.7 nM) and PKC beta II (IC50 = 5.9 nM) isozymes and was 76- and 61-fold selective for inhibition of PKC beta I and PKC beta II in comparison to PKC alpha, respectively. The additional analogues described in the series are also selective inhibitors of PKC beta. LY333531 (1) exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol ester (4 beta-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the series with ED50 values ranging from 7.5 to 0.21 microM. A comparison of the PKC isozyme and related ATP dependent kinase inhibition profiles is provided for the series and compared to the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series is compared with that of the kinase inhibitor staurosporine.

Detecting non-neutral heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence.

Natural selection, in the form of balancing selection or selective sweeps, can result in a decoupling of the amounts of molecular polymorphism and divergence. Thus natural selection can cause some areas of DNA sequence to have greater silent polymorphism, relative to divergence between species, than other areas. It would be useful to have a statistical test for heterogeneity in the polymorphism to divergence ratio across a region of DNA sequence, one that could identify heterogeneity greater than that expected from the neutral processes of mutation, drift, and recombination. The only currently available test requires that a region be arbitrarily divided into sections that are compared with each other, and the subjectivity of this division could be problematic. Here a test is proposed in which runs of polymorphic and fixed sites are counted, where a "run" is a set of one or more sites of one type preceded and followed by the other type. The number of runs is smaller than otherwise expected if polymorphisms are clumped together. By simulating neutral evolution and comparing the observed number of runs to the simulations, a statistical test is possible which does not require any a priori decisions about subdivision.

Bedside fluoroscopy to screen for simple extremity trauma in the ED.

Portable fluoroscopy units are commonly used by orthopedic surgeons to assist in fracture reduction and immobilization. The purpose of this study was to determine the diagnostic accuracy of bedside fluoroscopy performed by emergency department (ED) physicians to screen for simple extremity trauma. Eligible adult patients presenting to the ED with isolated injuries to distal extremities were evaluated prospectively over a 6-month study period. ED physicians independently performed fluoroscopy at the bedside, obtained real-time images (frontal, lateral, and oblique), and documented their initial interpretations. Patients then underwent routine diagnostic radiographs of the same areas. Fluoroscopic findings (real-time and photographs) were compared with the radiologists' final report. Ninety-two patients with 108 extremity injuries were enrolled in the study. Fractures were diagnosed fluoroscopically in 26 cases, for a sensitivity of 0.70. Of the 71 standard radiographs without a fracture, the fluoroscopic diagnosis was accurate in 66, for a specificity of 0.93. The overall diagnostic accuracy was 0.85, with a 95% confidence interval of .67 to 1.00. There were 11 false-negative fluoroscopic reports, involving the radius (3), distal tibia (3), metacarpals (2), fifth metatarsal (1), phalanx (1), and cuboid (1). These results suggest that bedside fluoroscopy lacks sufficient sensitivity to screen for simple extremity fractures in the ED.

Adaptive protein evolution at the Adh locus in Drosophila.

Proteins often differ in amino-acid sequence across species. This difference has evolved by the accumulation of neutral mutations by random drift, the fixation of adaptive mutations by selection, or a mixture of the two. Here we propose a simple statistical test of the neutral protein evolution hypothesis based on a comparison of the number of amino-acid replacement substitutions to synonymous substitutions in the coding region of a locus. If the observed substitutions are neutral, the ratio of replacement to synonymous fixed differences between species should be the same as the ratio of replacement to synonymous polymorphisms within species. DNA sequence data on the Adh locus (encoding alcohol dehydrogenase, EC 1.1.1.1) in three species in the Drosophila melanogaster species subgroup do not fit this expectation; instead, there are more fixed replacement differences between species than expected. We suggest that these excess replacement substitutions result from adaptive fixation of selectively advantageous mutations.

Comparative reactivity of 1-carba-1-dethiacephalosporins with cephalosporins.

Nine matched pairs of cephalosporins and their 1-carba-1-dethiacephalosporin analogues have been compared with regard to microbiological activity, beta-lactam carbonyl infrared absorption, and aqueous stability. In general the microbiological activity of the pairs of compounds were very similar across a broad range of bacteria. The infrared absorption bands for the beta-lactam carbonyls of the pairs indicated a general trend for the 1-carba-1-dethiacephalosporins to absorb at lower frequencies than the corresponding cephalosporins. All of the 1-carba-1-dethiacephalosporins did however present a striking stability enhancement over their cephalosporin counterparts at pH = 10 or 11 in water. This marked contrast of MIC similarity with the observed differences in chemical reactivity clearly demonstrates hydroxide ion catalyzed hydrolysis is not a good model for transpeptidase activity unless the compounds comprise a limited domain of structural type.

3-Quaternary ammonium 1-carba-1-dethiacephems.

A series of structurally unique 1-carba-1-dethiacephems is described. The structural stability of the 1-carba-1-dethiacephem nucleus was essential for the preparation of this series of 3-quaternary ammonium carbacephems. The known p-nitrobenzyl 7 beta-(phenoxyacetamido)- 3-[(trifluoromethyl)sulfonyl]oxy]-1-carba-1-dethia-3-cephem- 4-carboxylate served as both a quaternization substrate as well as a precursor to derivatives such as allyl 7 beta-[[2-[allyloxy)carbonyl]amino-4- thiazoly] (methoxyimino)acetyl]amino]-3-[(trifluoromethyl) sulfonyl] oxy]-1-carba-1-dethia-3-cephem-4-carboxylate. Quaternization of these enol triflates was accomplished either by dissolution in acetonitrile containing the base or by dissolution in the base, with or without warning to 50 degrees C. Bases nucleophilic enough to displace the triflate include a variety of substituted pyridines and N-methylimidazole. Deprotection then produced a very active series of 1-[7 beta-[(2-amino- 4-thiazolyl)(methoxyimino)acetyl]amino]-2-carboxy-8-oxo- 1-azabicyclo[4.2.0]oct-2-en-3-yl] quaternary ammonium hydroxide inner salts. These compounds were extremely potent antibacterials against a broad range of Gram-positive and -negative bacteria including constitutive cephalosporinase producers, such as Enterobacter cloacae. The compounds exhibit similar hydrolysis kinetics and pharmacokinetics to the analogous cephalosporin-3'-quaternary ammonium salts.

3-Sulfonyl-1-carba-1-dethiacephems.

The stability of the 1-carba-1-dethiacephalosporin framework has allowed the synthesis of a range of 3-sulfonyl-1-carba-1-dethiacephems unavailable for a variety of reasons in the cephem arena. The known p-nitrobenzyl 7 beta-(phenoxyacetamido)-3-[[(trifluoromethyl)sulfonyl]oxy]-1-carba -1- dethia-3-cephem-4-carboxylate served as a precursor to this series of compounds. Displacement of the enol triflate with various sulfinates in acetonitrile or DMF and deprotection of the intermediates led to 7 beta-[(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]- 3-[alkyl(aryl)sulfonyl]-1-carba-1-dethia-3-cephem-4-carboxyl ic acids. The 3-sulfonyl-1-carba-1-dethiacephems display potent activity against both Gram-positive and Gram-negative bacteria. The following MIC's (microgram/mL) for the 3-cyclopropyl sulfone are representative: Staphylococcus aureus = 4, Streptococcus pyogenes = 1, Haemophilus influenzae = 0.25, Escherichia coli = 0.03, Enterobacter cloacae = 0.25, Proteus rettgeri = 0.25. The excellent in vitro antibacterial activity of this series indicates the potential of the carbacephalosporin framework for exploring substituents which are unknown or which produce unstable cephems.

The politics of choice: roles of the medical profession under Nazi rule.

In 1933, when Adolf Hitler took power, the German medical community was faced with intense crisis and change. Because social processes become more clearly defined in times of crisis, the days of Nazi rule offer an excellent opportunity to examine health care and moral issues. This article describes historical events that illustrate physicians' and medical students' role in the political process. In addition, we detail four types of responses made by physicians and students: flight, conformism, individual resistance, and group resistance. We conclude that if the role of physicians is to aid and protect patients against disease or experimentation on humans, then he or she must maintain heightened political awareness in order to deal with social crises before they overwhelm any response.

Generation of oil-like pyrolyzates from organic-rich shales.

Pyrolyzates similar to natural crude oils were generated from organic-rich shales by hydrous pyrolysis. With this type of pyrolysis it is possible to make more sophisticated correlations between crude oils and their source rocks, evaluate the hydrocarbon potential of a source rock, and elucidate the variables involved in the natural oil-generating process.

Developing an integrated, competency-based medical technology program.

The authors describe a competency-based, integrated medical technology educational program currently being implemented at Weber State College in Ogden, Utah. Problems associated with the traditional three academic years plus one year clinical education program are described, as well as problems involved in creating new programs. This type of program may prove to be a major advance in the future of medical technology education.