Nasty Viruses, Costly Plasmids, Population Dynamics, and the Conditions for Establishing and Maintaining CRISPR-Mediated Adaptive Immunity in Bacteria

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses

Application of S-(+)-carvone in the synthesis of biologically active natural products using chemical transformations and bioconversions

S-(+)-Carvone, the major compound of caraway oil, was used as chiral starting material in the synthesis of biologically active natural compounds. Two annulation methodologies were explored to synthesize bicyclic intermediates from the monocyclic S-(+)-carvone. The bicyclic intermediates were further transformed into (+)-alpha-cyperone, a compound with anti-malaria activity, (-)-polygodial, an insect-antifeedant, a decalol that inhibits the cholesterol biosynthesis and (-)-Ambrox(R), a commercially interesting olfactory compound. S-(+)-Carvone was used as substrate for bioconversions by selected micro-organisms. S-(+)-Carvone was reduced predominantly to both dihydrocarvones and to neo-iso-dihydrocarveol. Sensitivity of the micro-organisms for S-(+)-carvone and some of the products prevented yields exceeding 0.35 g/l in batch-cultures. The fungus Trychoderma pseudokoningii had the highest yield percentage of neo-isodihydrocarveol

Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis

The endoperoxide sesquiterpene lactone artemisinin and its derivatives are a promising new group of drugs against malaria. Artemisinin is a constituent of the annual herb Artemisia annua L. So far only the later steps in artemisinin biosynthesis - from artemisinic acid - have been elucidated and the expected olefinic sesquiterpene intermediate has never been demonstrated. In pentane extracts of A. annua leaves we detected a sesquiterpene with the mass spectrum of amorpha-4,11-diene. Synthesis of amorpha-4,11-diene from artemisinic acid confirmed the identity. In addition we identified several sesquiterpene synthases of which one of the major activities catalysed the formation of amorpha-4,11-diene from farnesyl diphosphate. This enzyme was partially purified and shows the typical characteristics of sesquiterpene syntheses, such as a broad pH optimum around 6.5-7.0, a molecular mass of 56 kDa, and a K-m of 0.6 mu M. The structure and configuration of amorpha-4,11-diene, its low content in A. annua and the high activity of amorpha-4,11-diene synthase all support that amorpha-4,11-diene is the likely olefinic sesquiterpene intermediate in the biosynthesis of artemisinin. (C) 1999 Elsevier Science Ltd. All rights reserved

Qualitative observation of left ventricular multiphasic septal motion and septal-to-lateral apical shuffle predicts left ventricular reverse remodeling after cardiac resynchronization therapy

A multiphasic septal motion and typical septal-to-lateral apical shuffle of the left ventricle can be observed echocardiographically in some patients with left branch bundle block. The relation of both with left ventricular (LV) dyssynchrony according to tissue Doppler and LV reverse remodeling after cardiac resynchronization therapy was investigated. Fifty-three patients (37 men; age 68 ± 8 years) with ischemic (n = 26) or idiopathic (n = 27) cardiomyopathy, baseline QRS duration 171 ± 30 ms, LV ejection fraction 21 ± 7%, and LV end-diastolic volume 257 ± 91 ml were studied. LV dyssynchrony using tissue Doppler was considered present if the SD of the interval between QRS and onset of systolic velocity of 6 basal LV segments was >20 ms. Shuffle was evaluated visually independently by 5 cardiologists and considered present if observed in =1 view. LV reverse remodeling, defined as LV end-systolic volume decrease =10%, was observed in 37 patients (70%) after 3 months of CRT. Sensitivity and specificity of either shuffle or multiphasic septal motion for all 5 observers (range 90% to 97% and 67% to 83%, respectively) were found to predict LV dyssynchrony. To predict LV reverse remodeling, sensitivity and specificity from 87% to 92% and 69% to 81% were observed, respectively. In conclusion, the qualitative observation of a typical shuffle or multiphasic septal motion predicts LV dyssynchrony and LV reverse remodeling adequately