Lactic acid bacteria as prime candidates for codon optimization

In species having a strong correlation of expressivity and codon bias it has been shown that heterologous expression can be optimized by changing codons of the introduced gene towards the set of codons that the host organism naturally uses in its highly expressed genes. Even though two lactic acid bacteria are fully sequenced, there are no reports on attempts of codon optimization in the literature. In this report it is demonstrated that codons used in highly expressed genes tend to differ from the codons in lowly expressed genes, and that there is a strong correlation of codon bias and empirical expressivity (codon adaptation index) in Lactococcus lactis and Lactobacillus plantarum. This strongly suggests that codon optimization strategies could be applied to expression systems with lactic acid bacteria as producer strains. A good example of a candidate for codon optimization is the mouse interleukin-2 gene, which in its natural form has an extremely low codon adaptation index for expression in Lc. lactis.     

Binding,internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts

Binding, internalization, and degradation of ¹²⁵I-labeled, antiproliferative, or nonantiproliferative heparan sulfate by human embryonic lung fibroblasts was investigated. Both L-iduronate-rich, antiproliferative heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan sulfate types were bound with approximately the same affinity to one high-affinity site (K_d approximately 10⁻⁸ M) and to one (K_d approximately 10⁻⁶ M), respectively. Results of Hill-plot analysis suggested that the two sites are independent. Competition experiments with unlabeled glycosaminoglycans indicated that the binding sites had a selective specificity for sulfated, L-iduronate-rich heparan sulfate. Dermatan sulfate, which is also antiproliferative, was weakly bound to the cells. The antiproliferative effects of heparan and dermatan sulfate appeared to be additive. Hence, the two glycosaminoglycans probably exert their effect through different mechanisms. At concentrations above 5 μg/ml (approximately 10⁻⁷ M), heparan sulfate was taken up by human embryonic lung fibroblasts, suggesting that the low-affinity site represents an endocytosis receptor. The antiproliferative effect of L-iduronate-rich heparan sulfate species was also exerted at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran sulfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural analysis of the inactive and active heparan sulphate preparations indicated that although sulphated L-iduronate appears essential for antiproliferative activity, it is not absolutely required for binding to the cells. Degradation of internalized heparan sulfate was analyzed by polyacrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiproliferative one was only marginally affected. J. Cell. Biochem. 64:595–604. © 1997 Wiley-Liss, Inc.     

Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.     

Digital transformation—life cycle assessment of digital services,multifunctional devices and cloud computing

The International Journal of Life Cycle Assessment -     

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA _I ^Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA _I ^Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.Abbreviations IMT and imt (imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene - met-tRNA _I ^Met methionylated initiator methionine transfer RNA - eIF-2 eukaryotic initiation factor two - GTP guanosine 5-triphosphate The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)     

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1–2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.     

Prevalence of the genes encoding propionicin T1 and protease-activated antimicrobial peptide and their expression in classical propionibacteria

The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene (pamA) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene (pctA) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA-containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame (orf2) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.     

Induction of heavy metal binding groups by dexamethasone and Zn in cultured Chinese hamster ovary cells

Cd binding capacity and pulse polarography were used to study the inducibility of sulfhydryl groups in cultured Chinese hamster ovary cells (wild type and a Cd-resistant mutant) in response to dexamethasone (dex) and Zn. Evidence is presented that both the wild type and the mutant responded to dex and Zn treatment by induction of sulfhydryl groups. In wild type for Zn and dex as well as in the mutant for dex, this induction seems to be in the form of sulfhydryls attached to particulate or membrane fractions in the cells. For Zn in the Cd-resistant mutant the induction was in the form of metallothionein.