Total Reconstitution of Functionally Active 50S Ribosomal Subunits from Escherichia coli

Total reconstitution of 50S subunits from E. coli was achieved by a two-step incubation procedure. In the first step, 23S RNA, 5S RNA, and the total proteins from 50S subunits were incubated for 20 min at 40 degrees in the presence of 4 mM Mg(++) and 400 mM NH(4)Cl. In the second step, the Mg(++) concentration was raised to 20 mM and the incubation was performed for 90 min at 50 degrees . No requirement for 30S subunits or other components (e.g., polyamine) was found. The reconstituted particle has the same sedimentation coefficient as the native 50S subunit and is highly active in protein synthesis with natural (R17 RNA) and artificial [poly(U)] messengers as well as in tests for peptidyltransferase (fragment assay) and for binding of antibiotics (chloramphenicol).     

Identification of the Chloramphenicol-Binding Protein in Escherichia coli Ribosomes by Partial Reconstitution

50S-derived cores were prepared by treatment of 50S subunits with 0.4 M Licl (0.4c core) and 0.8 M Licl (0.8c core), respectively. 0.4c cores bind chloramphenicol whereas 0.8c cores do not. The split proteins obtained during the transitions 0.4c --> 0.8c were separated by DEAE-cellulose chromatography and Sephadex G-100 gel filtration. Reconstitution experiments with the fractionated proteins demonstrated that protein L16 is involved in chloramphenicol binding.In contrast to chloramphenicol, the CACCA-(N-acetyl-leucyl) fragment is bound by the 0.8c core, i.e., this core contains the intact p-site moiety of the peptidyltransferase center.Puromycin can inhibit chloramphenicol binding completely. In the concentration range tested (up to 20 mM) the trinucleotide CCA inhibits chloramphenicol binding as effectively as puromycin, whereas an aminoacid mixture shows no inhibition. It is concluded that chloramphenicol acts exclusively on the a-site part of the peptidyltransferase center interfering with the binding of the last two or three nucleotides (3' end) of aminoacyl-tRNA.     

Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors

Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. CONCLUSION: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.     

Current Smokers Develop More Posterior Myocardial Infarctions Probably Due to Increased Tendency to Thrombosis

This investigation was carried out to determine whether smokers developed smaller infarcts as assessed by peak enzyme levels and also to what extent smoking could modify infarct localization. The study included 753 patients, of whom 351 had no history of previous coronary heart disease (CHD) (angina pectoris and/or myocardial infarction (MI)). The investigation was designed as an exposed (smoking) versus non-exposed (non-smoking) cohort study. Outcome was infarct size, posterior versus non-posterior MI and non-Q-wave versus Q-wave infarcts. In the total cohort of patients, 312 (41%) were smokers, the corresponding number in the restricted cohort of patients without a previous CHD (CHD-0-pts) was 169 (48%). Smokers were younger than non-smokers, and more of them were males. It was found that infarct size was similar in smokers and in non-smokers (crude and adjusted effects). Crude effects showed that smokers developed significantly more posterior infarcts than non-smokers; odds ratio (OR) for developing a posterior MI was 1.95 (2p < 0.001) (all patients) and 2.34 (2< 0.001) (CHD-0-pts), respectively. After adjusting for confounders (logistic regression model), OR in the two groups was 1.24 (2p = 0.256) and 1.95 (2p = 0.01), respectively. The study shows that current smokers were younger, and indicates that in those without a previous CHD, significantly more of them developed a posterior MI.     

Time-trends in method-specific suicide rates compared with the availability of specific compounds. The Danish experience

Restriction of means for suicide is an important part of suicide preventive strategies in different countries. All suicides in Denmark between 1970 and 2000 were examined with regard to method used for suicide. Overall suicide mortality and method-specific suicide mortality was compared with official information about availability of medical compounds (barbiturates, benzodiazepines, analgesics, antidepressants) and carbon monoxide in vehicle exhaust and household gas. Restrictions on the availability of carbon monoxide, barbiturates and dextropropoxyphen was associated with a decline in the number of suicides by self-poisoning with these compounds. Restricted access occurred concomittantly with a 55% decrease in suicide rate.     

Microevolution and patterns of dissemination of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans

The natural history, microevolution, and patterns of interindividual transmission and global dissemination of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans were studied by population genetic analysis. The JP2 clone is strongly associated with aggressive periodontitis in adolescents of African descent and differs from other clones of the species by several genetic peculiarities, including a 530-bp deletion in the promoter region of the leukotoxin gene operon, which results in increased leukotoxic activity. Multilocus sequence analysis of 82 A. actinomycetemcomitans strains, 66 of which were JP2 clone strains collected over a period of more than 20 years, confirmed that there is a clonal population structure with evolutionary lineages corresponding to serotypes. Although genetically highly conserved, as shown by alignment of sequences of eight housekeeping genes, strains belonging to the JP2 clone had a number of point mutations, particularly in the pseudogenes hbpA and tbpA. Characteristic mutations allowed isolates from individuals from the Mediterranean area and from West Africa, including the Cape Verde Islands, to be distinguished. The patterns of mutations indicate that the JP2 clone initially emerged as a distinct genotype in the Mediterranean part of Africa approximately 2,400 years ago and subsequently spread to West Africa, from which it was transferred to the American continents during the transatlantic slave trade. The sustained exclusive colonization of individuals of African descent despite geographical separation for centuries suggests that the JP2 clone has a distinct host tropism. The colonization of family members by JP2 clone strains with unique point mutations provides strong evidence that there is intrafamilial transmission and suggests that dissemination of the JP2 clone is restricted to close contacts.     

Codon-anticodon interaction at the ribosomal P (peptidyl-tRNA) site

A method for binding tRNA to ribosomes, introduced by Watanabe [Watanabe, S. (1972) J. Mol. Biol. 67, 443-457], permits nonenzymatic binding of N-acetyl-Phe-tRNA(Phe) to either the ribosomal aminoacyl-tRNA (A) or peptidyl-tRNA (P) site with almost 100% specificity. We used this method to analyze a possible codon-anticodon interaction at the P site for NH(2)-blocked aminoacyl-tRNA and deacylated tRNA. N-Acetyl-Phe-tRNA(Phe) bound only to the P site of poly(U)-programmed 70S ribosomes, not to poly(A)-programmed ribosomes. The reverse mRNA dependence was found for N-acetyl-Lys-tRNA(Lys). A series of purified deacylated tRNAs was analyzed in the poly(U) and poly(A) system for abilities to block P-site binding of N-acetyl-aminoacyl-tRNA and to direct the N-acetyl-aminoacyl-tRNA to the A site. Only the cognate tRNA was as effective as the bulk tRNA at a concentration of less than 1/20th that of bulk tRNA. tRNAs whose corresponding codons are identical or similar (same base character) in the first two codon positions showed a low but significant effect. The other noncognate tRNAs were unable to direct the NH(2)-blocked aminoacyl-tRNAs to the A site. Chlortetracycline interfered neither with the P-site binding of NH(2)-blocked aminoacyl-tRNA nor with the effects of deacylated tRNAs. Furthermore, the translocation blocker viomycin affected neither the binding to the A site nor that to the P site. These effects of both antibiotics indicate that both kinds of tRNA do not bind transiently in the A site before filling the P site and that codon-anticodon interaction takes place at the P site.     

Clade extinction appears to balance species diversification in sister lineages of Afro-Oriental passerine birds

Recent analyses suggest that the number of species in a clade often increases rapidly at first, but that diversification subsequently slows, apparently as species fill ecological space. Support for diversity dependence comes largely from the failure of species richness to increase with clade age in some analyses of contemporary diversity. However, clades chosen for analysis generally are named taxa and thus are not selected at random. To avoid this potential bias, we analyzed the numbers of species and estimated ages of 150 pairs of sister clades established by dispersal of ancestral species between the Oriental and African biogeographic regions. The observed positive exponential relationship between clade size and age suggests that species diversify within clades without apparent limit. If this were true, the pattern of accumulation of sister-clade pairs with increasing age would be consistent with the random decline and extinction of entire clades, maintaining an overall balance in species richness. This “pulse” model of diversification is consistent with the fossil record of most groups and reconciles conflicting evidence concerning diversity dependence of clade growth.The close relationship between local species richness and characteristics of the physical environment supports the existence of ecological constraints on species coexistence mediated through competition and other interactions (1–3), although historical influences on diversity sometimes parallel gradients in the physical environment (4–6). If species richness were limited as ecological space filled, one would expect the net rate of species production to slow and the number of species in a clade to level off as species richness approached ecological constraints.Evidence for such “diversity-dependent diversification” consists mostly of (i) nonrandom concentrations of branch points (speciation events) toward the origin of a clade (7–12) and (ii) independence of the number of species and clade age in comparisons among clades (11–14). However, clades included in such analyses often are not randomly chosen. In particular, small clades may be ignored because they do not command interest, and large, older clades are often passed over because of incomplete sampling (15, 16). Moreover, most phylogenetic analyses of diversification include species in named higher taxa rather than clades that have diversified within particular regions.Further support for diversity limits comes from the fossil records of many higher taxa, which exhibit long-term stability in number of species (17–19). It is also clear that species and entire clades continually replace each other through time, and the dynamics of this process appear to include the decline and extinction of evolutionary groups as a component of the local and regional regulation of the number of coexisting species (20–25).We take advantage of the sister relationships of clades of passerine birds in two major biogeographic regions—tropical southern Asia [Oriental (OR)] and the continent of Africa [African (AF)]—to examine the independent diversification of sister clades of known stem ages, selected only because one of the ancestors had dispersed between the two continents at some time in the past. Movement of species between these regions occurred either over water across the Indian Ocean, possibly using island stepping-stones, or through the Arabian Peninsula during periods of suitable environment. Each dispersal event defines the origin of a pair of same-aged sister clades in the two regions.If each diversifying clade filled a certain part of ecological space to a carrying capacity for species, after which diversification slowed, the sizes of clades filling this space would level off over time (26). Furthermore, the number of species per sister clade, particularly among older clades that have filled ecological space, might be correlated between regions, with species richness reflecting the ecological space available to each of the sister lineages (27). Finally, for those dispersal events whose directionality can be inferred, the rate of diversification should be higher, leading to larger clade size compared between sister lineages, in the newly colonized region, which initially would have fewer close (and ecologically similar) relatives of the ancestral species.We include all species of passerine birds (Passeriformes) and, separately, species in nonpasserine orders, of small, terrestrial birds, in the Oriental and African biogeographic regions (SI Appendix, section S1). The passerine avifauna of these regions accumulated from several sources over most of the Cenozoic Era (28, 29), with an old Gondwanan clade of suboscine passerines diversifying in tropical Africa and southern Asia early in the Tertiary, followed by radiations of core corvoid and passerid oscine passerines out of Australia during the early to mid-Cenozoic, through Wallacea to southern Asia, or directly across the Indian Ocean to Africa (30, 31). Thus, the diversification of sister clades in the African and Oriental regions takes place against a background of an increasing number of lineages of modern passerine birds as a whole within the region. Nonpasserine orders of terrestrial birds are likely to have diversified earlier within these regions (32) and perhaps were replaced to some extent by the passerines.Characteristics of a sample of clades include the distribution of node ages and the relationship between clade size and stem age, as well as the distribution of clade sizes regardless of age, including the proportion of clades that contain a single species. Any process-based model of diversification should be judged by how well it reproduces these characteristics. We use these criteria to evaluate simulations in which we attempt to reconstruct the underlying diversification process.     

Effect of simvastatin on coronary lesion site remodeling: a serial intravascular ultrasound study

BACKGROUND: Direct evidence of coronary artery remodeling can be derived only from serial changes in the external elastic membrane (EEM) and plaque area. The aim of the study was to assess the effect of simvastatin on coronary remodeling in serial intravascular ultrasound (IVUS) studies. METHODS: In 39 male patients ECG-triggered transducer pullback IVUS was performed at baseline, after 3 months on a lipid-lowering diet (control period), and after another 12 months of simvastatin 40 mg/day. The lesion site was the image slice with maximum plaque burden at 3 months. RESULTS: Absolute changes in the EEM area correlated significantly with changes in plaque area during the control period [B = 0.966, r = 0.792 (95% CI 0.71-1.22); p < 0.001] and during simvastatin treatment [B = 0.945, r = 0.822 (95% CI 0.73-1.16); p < 0.001], but there was no significant difference in the slope (delta EEM/delta plaque) between the two time intervals. After 12 months of simvastatin, there was a significant reduction in the lesion EEM area of 4.6% (p = 0.006) and in the lesion plaque area of 5.9% (p < 0.001), but there was no change in reference measurements. As a result, the remodeling index was reduced by simvastatin from 1.01 +/- 0.12 to 0.95 +/- 0.09 (p < 0.001). CONCLUSION: Simvastatin decreases the remodeling index by reducing lesion, but not reference plaque and EEM area. However, simvastatin does not affect direct evidence of remodeling (delta EEM/delta plaque) obtained using serial IVUS studies.