Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1 beta

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.     

Shrinkage of the non-malignant prostate gland volume after receiving incidental radiotherapy for rectal cancer

BackgroundThe purpose of this study was to assess the impact of coincidental radiotherapy on the volume of the non-malignant prostate gland in rectal cancer patients treated with neo-adjuvant radiotherapy.Materials and methodsIn this retrospective analysis, thirty male patients with rectal cancer who had neoadjuvant radiotherapy met the inclusion criteria. These patients had pre-treatment magnetic resonance imaging (MRI) and at least one post-treatment MRI of the pelvis and the whole of their prostate volume received the full prescribed radiotherapy dose; 45 Gy in 25 fractions (n = 22), 45 Gy in 20 fractions (n = 4) and 25 Gy in 5 fractions (n = 4).ResultsThe median age of this patient cohort was 66 years (range: 30–87). With a median interval between pre-treatment MRI and first MRI post-treatment of 2 months (range: 1–11), the mean prostate volume reduced from 36.1 cm³ [standard deviation (SD) 14.2] pre-radiotherapy to 31.3 cm³ (SD 13.0) post radiotherapy and this difference was significant (p = 0.0004).ConclusionRadiotherapy may cause shrinkage in volume of normal (non-malignant) prostate. Further research is required in this field, since these results may be of some comfort to men contemplating the consequences of radiotherapy on their quality of life. The authors suggest recording flow-rate and international prostate symptom score (IPSS) during rectal radiotherapy as a next step.     

Expansion of Microsatellites on Evolutionary Young Y Chromosome

Sex chromosomes are an ideal system to study processes connected with suppressed recombination. We found evidence of microsatellite expansion, on the relatively young Y chromosome of the dioecious plant sorrel (Rumex acetosa, XY1Y2 system), but no such expansion on the more ancient Y chromosomes of liverwort (Marchantia polymorpha) and human. The most expanding motifs were AC and AAC, which also showed periodicity of array length, indicating the importance of beginnings and ends of arrays. Our data indicate that abundance of microsatellites in genomes depends on the inherent expansion potential of specific motifs, which could be related to their stability and ability to adopt unusual DNA conformations. We also found that the abundance of microsatellites is higher in the neighborhood of transposable elements (TEs) suggesting that microsatellites are probably targets for TE insertions. This evidence suggests that microsatellite expansion is an early event shaping the Y chromosome where this process is not opposed by recombination, while accumulation of TEs and chromosome shrinkage predominate later.     

Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics

Background: The evolution and biology of rDNA have interested biologistsfor many years, in part, because of two intriguing processes:(1) nucleolar dominance and (2) sequence homogenization. Wereview patterns of evolution in rDNA in the angiosperm genusNicotiana to determine consequences of allopolyploidy on theseprocesses. Scope: Allopolyploid species of Nicotiana are ideal for studying rDNAevolution because phylogenetic reconstruction of DNA sequenceshas revealed patterns of species divergence and their parents.From these studies we also know that polyploids formed overwidely different timeframes (thousands to millions of years),enabling comparative and temporal studies of rDNA structure,activity and chromosomal distribution. In addition studies onsynthetic polyploids enable the consequences of de novo polyploidyon rDNA activity to be determined. Conclusions: We propose that rDNA epigenetic expression patterns establishedeven in F₁ hybrids have a material influence on the likely patternsof divergence of rDNA. It is the active rDNA units that arevulnerable to homogenization, which probably acts to reducemutational load across the active array. Those rDNA units thatare epigenetically silenced may be less vulnerable to sequencehomogenization. Selection cannot act on these silenced genes,and they are likely to accumulate mutations and eventually beeliminated from the genome. It is likely that whole silencedarrays will be deleted in polyploids of 1 million years of ageand older.     

Performance of WHO Angina Questionnaire in measuring burden of coronary heart disease in human isolate populations

Isolated human populations represent good candidates for studying genetic and environmental causes of common complex diseases because of their decreased genetic and environmental diversity. The possibility of inexpensive and reliable detection of disease prevalence in such populations is therefore of considerable importance, as comprehensive routine health data and disease registries are rarely available in these populations. In this study, we validated the performance of the WHO Rose Angina Questionnaire (RQ) in measuring the burden of coronary heart disease (CHD) in 9 settlements in these Croatian Adriatic islands. CHD was defined as myocardial infarction (MI) diagnosed by a specialist in the local general hospital, or angina pectoris (AP) by a local general practitioner (GP). The true prevalence of CHD in 1,001 adult persons was 10.5%. The results of the RQ screening based on the first 3, 5 and 6 questions were compared with medical record of CHD. Increasing the number of RQ questions from 3 to 6 resulted in decreasing test sensitivity (from 59.0% to 30.5%) and increasing test specificity (from 86.3% to 93.0%) in the prediction of true CHD status. CHD prevalence was overestimated by 76% when subset of the first 3 questions of RQ was used and by 25% when the first 5 questions were used. However, it was underestimated by 10% when the first 6 questions were used. We conclude that RQ is a useful screening method for measuring burden of CHD in isolate human populations, and that the result based on the first 6 questions is a good approximation of the true CHD prevalence in the population, although it should be considered a slight underestimate.