The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli. A mutagenesis study

Methods of in vitro mutagenesis were employed to determine the importance of individual nucleotides within the ribosomal RNAs for the structure and function of E. coli ribosomes. A series of defined nucleotides in the genes for the 5 S and 16 S RNA were altered by transition and transversion mutations using either oligonucleotide-directed or bisulfite-catalyzed mutation procedures. Plasmids harbouring the mutated rRNA genes were expressed and the ribosomes containing such altered RNAs were investigated for impairments in RNA-protein interaction assembly and mRNA-coded tRNA binding.     

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl)glutathione and N-ethylmaleimide showed intermediate inhibitory effects.     

Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns

Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi‐product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 10⁶ cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Biotechnol. Bioeng. 2013; 110: 1376–1385. © 2012 Wiley Periodicals, Inc.     

Penicillin-binding protein 2a of Streptococcus pneumoniae: expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a, designated GST-PBP2a*, was expressed almost exclusively as inclusion bodies. Using a combination of high- and low-speed centrifugation, large amounts of purified inclusion bodies were obtained. These purified inclusion bodies were refolded into a soluble and enzymatically active enzyme using a single-step refolding method consisting of solubilization of the inclusion bodies with urea and direct dialysis of the solubilized preparations. Using these purification and refolding methods, approximately 37 mg of soluble GST-PBP2a* protein was obtained from 1 liter of culture. The identity of this refolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a beta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicillin G. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were obtained and shown to be enzymatically active.     

Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation

We have studied the relationship of valency of CD3 stimulation and modulation of the CD3 receptor complex with biochemical and proliferative responses of T cells. Anti-CD3 Fab, as well as F(ab')2 and whole antibody caused rapid modulation of the CD3 antigen, whereas anti-CD3 conjugated to Sepharose did not. In the absence of monocytes, T cells stimulated with anti-CD3 Fab, F(ab')2, or F(ab')2-Sepharose showed differences in their ability to respond to second signals given by PMA, IL 1, IL 2, or antibodies to Tp67 and Tp44. None of the anti-CD3 signals alone caused resting T cells to produce IL 2, and only the Sepharose-bound anti-CD3 F(ab')2 caused T cells to express high levels of functional IL 2 receptors. Anti-CD3 F(ab')2-Sepharose-stimulated T cells produced IL 2 and proliferated in response to each of the second signals. Because anti-CD3-Sepharose did not cause modulation of the CD3 antigen, the ability of the Sepharose-bound antibody to induce T cells to express IL 2 receptors and to respond to individual second signals may be related to lack of modulation rather than valency of binding. Anti-CD3 Fab-stimulated T cells responded to PMA but required combinations of other second signals. T cells stimulated with unmodified anti-CD3 antibody or F(ab')2 fragments responded to PMA but did not respond to any other second signals alone or in combination. Stimulations that resulted in modulation (i.e., anti-CD3 whole antibody, anti-CD3 F(ab')2, or anti-CD3 Fab fragments) caused an increase in cytoplasmic calcium levels in resting T cells but blocked proliferation of T cells in response to mitogenic lectins or CD2 stimulation. Anti-CD3 F(ab')2 on Sepharose, however, did not block T cell proliferation. Whole bivalent anti-CD3 antibody or F(ab')2 fragments, but not monovalent Fab fragments, caused a rapid translation of protein kinase C activity from cytosol to membrane in the Jurkat T cell line. Because all of these modulate the receptor, these data indicate that the functional difference between monovalent and bivalent binding to CD3 is related to antibody valency and not to antigenic modulation. The use of Fab anti-CD3 stimulation that requires combinations of second signals for proliferation allowed an analysis of the functional relationships between IL 1, anti-Tp67, and anti-Tp44.     

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67

Cryptococcus neoformans is an opportunistic human pathogen that causes life‐threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI‐MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal–host relationship is discussed.     

Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide

Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.     

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.     

Yeast as a model for studying human neurodegenerative disorders

Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.