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PubMed2025

Robust and highly efficient transformation method for a minimal mycoplasma cell.

UNLABELLED: Mycoplasmas have been widely investigated for their pathogenicity, as well as for genomics and synthetic biology. Conventionally, transformation of mycoplasmas was not highly efficient, and due to the low transformation efficiency, large amounts of DNA and recipient cells were required for that purpose. Here, we report a robust and highly efficient transformation method for the minimal cell JCVI-syn3B, which was created through streamlining the genome of Mycoplasma mycoides. When the growth states of JCVI-syn3B were examined in detail by focusing on such factors as pH, color, absorbance, colony forming unit, and transformation efficiency, it was found that the growth phase after the lag phase can be divided into three distinct phases, of which the highest transformation efficiency was observed during the early exponential growth phase. Notably, the transformation efficiency of up to 4.4 × 10-2 transformants per cell per microgram of plasmid DNA was obtained. A method to obtain several hundred to several thousand transformants with less than 0.2 mL of culture with approximately 1 × 107-108 cells and 10 ng of plasmid DNA was developed. Moreover, a transformation method using a frozen stock of transformation-ready cells was established. These procedures and information could simplify and enhance the transformation process of minimal cells, facilitating advanced genetic engineering and biological research using minimal cells. IMPORTANCE: Mycoplasmas are parasitic and pathogenic bacteria for many animals. They are also useful bacteria to understand the cellular process of life and for bioengineering because of their simple metabolism, small genomes, and cultivability. Genetic manipulation is crucial for these purposes, but transformation efficiency in mycoplasmas is typically quite low. Here, we report a highly efficient transformation method for the minimal genome mycoplasma JCVI-syn3B. Using this method, transformants can be obtained with only 10 ng of plasmid DNA, which is around one-thousandth of the amount required for traditional mycoplasma transformations. Moreover, a convenient method using frozen stocks of transformation-ready cells was established. These improved methods play a crucial role in further studies using minimal cells.

Transformation, Bacterial↗
PubMed2002

Insights into the regulation of gastric acid secretion through analysis of genetically engineered mice.

The regulation of acid secretion in the stomach involves a complex network of factors that stimulate secretion in response to the ingestion of a meal and maintain homeostasis of gastric pH. Genetically engineered mouse models have provided a new opportunity to investigate the importance and function of specific molecules and pathways involved in the regulation of acid secretion. Mouse mutants with disruptions in the three major stimulatory pathways for acid secretion in parietal cells, gastrin, histamine, and acetylcholine, have been generated. Disruption of the gastrin pathway results in a major impairment in both basal and induced acid secretion. Histamine and acetylcholine pathway mutants also have significant alterations in acid secretion, although the impairment does not appear to be as severe as in gastrin pathway mutants, perhaps due in part to the hypergastrinemia that occurs. Mice with a disruption in the somatostatin pathway have increased gastric acid secretion, which confirms an important negative regulatory role for this factor. This review discusses these genetically engineered mouse models, as well as others, that provide insight into the complex regulation of in vivo gastric acid secretion. The regulation of growth and cellular morphology of the stomach in these mouse models is also presented. In addition, transgene promoters that are expressed in the gastric epithelium are discussed because these promoters will be important tools to alter cellular physiology in new mouse models in the future.

Acetylcholine↗
PubMed2002

Biologic liver support: optimal cell source and mass.

Hepatic support is indicated in acute liver failure (ALF) patients to foster liver regeneration, or until a liver becomes available for orthotopic liver transplantation (OLT), in primary non function of the transplanted liver, and hopefully in chronic liver disease patients affected by ALF episodes, in whom OLT is not a therapeutic option. The concept of bioartificial liver (BAL) is based on the assumption that only the hepatocytes can perform the whole spectrum of biotransformation functions, which are needed to prevent hepatic encephalopathy, coma and cerebral edema. Among others, two important issues are related to BAL development: 1) the choice of the cellular component; 2) the cell mass needed to perform an adequate BAL treatment. Primary hepatocytes, of human or animal origin, should be considered the first choice because they express highly differentiated functions. Accordingly, a minimal cell mass corresponding to 10% of a human adult liver, i.e. 150 grams of freshly isolated, > or = 90% viable hepatocytes should be used. When 4 degrees C cold-stored or cryopreserved hepatocytes are used, the cellular mass should be increased because of a drop in cell viability and function. In case of hepatoma-derived cells, cultured cell lines or engineered cells, an adequate functional cell mass should be used, expressing metabolic and biotransformation activities comparable to those of primary hepatocytes. Finally, the use of porcine hepatocytes or other animal cells in BAL devices should be presently directed only to ALF patients as a bridge treatment to OLT, because of potential transmission of animal retrovirus and prions which may potentially cause major pandemics.

Animals↗
PubMed2006

PIN-G--a novel reporter for imaging and defining the effects of trafficking signals in membrane proteins.

BACKGROUND: The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells. RESULTS: Consequently, we have engineered a mammalian expression vector encoding a novel trafficking reporter--pIN-G--consisting of a simple, type I integral protein bearing permissive intra/extracellular cloning sites, green fluorescent protein (GFP), cMyc and HA epitope tags. Fluorescence imaging, flow cytometry and biochemical assays of transfected HEK293 cells, confirm the size, topology and surface expression of PIN-G. Moreover, a pIN-G fusion construct, containing a Trans-Golgi Network (TGN) targeting determinant, internalises rapidly from the cell surface and localises to the TGN. Additionally, another PIN-G fusion protein and its mutants reveal trafficking determinants in the cytoplasmic carboxy terminus of Kv1.4 voltage-gated potassium channels. CONCLUSION: Together, these data indicate that pIN-G is a versatile, powerful, new reporter for analysing signals controlling membrane protein trafficking, surface expression and dynamics.

Cell Line↗
PubMed2006

Non-NMDA-type glutamate receptors are essential for maturation but not for initial assembly of synapses at Drosophila neuromuscular junctions.

The assembly of glutamatergic postsynaptic densities (PSDs) seems to involve the gradual recruitment of molecular components from diffuse cellular pools. Whether the glutamate receptors themselves are needed to instruct the structural and molecular assembly of the PSD has hardly been addressed. Here, we engineered Drosophila neuromuscular junctions (NMJs) to express none or only drastically reduced amounts of their postsynaptic non-NMDA-type glutamate receptors. At such NMJs, principal synapse formation proceeded and presynaptic active zones showed normal composition and ultrastructure as well as proper glutamate release. At the postsynaptic site, initial steps of molecular and structural assembly took place as well. However, growth of the nascent PSDs to mature size was inhibited, and proteins normally excluded from PSD membranes remained at these apparently immature sites. Intriguingly, synaptic transmission as well as glutamate binding to glutamate receptors appeared dispensable for synapse maturation. Thus, our data suggest that incorporation of non-NMDA-type glutamate receptors and likely their protein-protein interactions with additional PSD components triggers a conversion from an initial to a mature stage of PSD assembly.

Animals↗
PubMed1995

Considerations for the use of cytokine-secreting tumor cell preparations for cancer treatment.

Limited efficacy of chemotherapy in most solid tumors has revived interest in immunotherapeutic approaches for cancer. One novel form of immunotherapy is the use of cancer vaccines consisting of tumor cells genetically engineered to secrete cytokines. The rationale for this immunization strategy is based on the existence of tumor-specific antigens, on the importance of the cellular arm of the immune system in mediating an effective antitumor response, and on the role of cytokines in regulating the cellular immune response. Such tumor vaccines showed considerable promise in various animal models and induced potent antitumor immunity in the host, which led to regression of established tumors and, moreover, produced immunological memory protecting animals from a subsequent tumor challenge at a distant site. Translated to the human patient, this implies that genetically modified tumor vaccines may be able to eradicate or reduce existing tumor deposits to subclinical levels as well as provide long-term protection from regrowth of tumor cells. This report will review and discuss the concept and rationale for the use of cytokine-secreting tumor vaccines for the treatment of human malignancies.

Animals↗
PubMed2000

[Gene cloning and expression of prolyl endopeptidase from Aeromonas punctata].

Prolyl endopeptidase gene was cloned from Aeromonas punctata subsp. Punctata(ST-78-3-3) using activity screening method and the 3.3 kb DNA fragment containing PEP gene was sequenced. DNA sequence from 20-2092 bp was ORF region coding 690 amino acids of prolyl endopeptidase. It was a new PEP gene through gene search. The genetic engineered E. coli BL21/pGEM-PEP overexpressing recombinant Aeromonas punctata prolyl endopeptidase(apPEP) was constructed. Cultured in YH medium, the expressed apPEP was about 30% of total cellular protein, the activity was 112 times more than that of wild strain. Expressed apPEP was mainly soluble intracellular protein, about 5% of apPEP was secreted to medium. Non-reduced SDS-PAGE analysis showed it's monomer with molecular weight about 76 kD, which corresponded with prediction by gene sequence. Recombinant apPEP was purified after tube culture, the purity reached 90% and specific activity was 67 U/mg.

Aeromonas↗
PubMed1991

Design of site-specific protein drugs.

The ability to clone and express proteins at high levels of abundance and purity has led to their increasing consideration as therapeutic agents. The use of various protein engineering and recombinant DNA techniques has enabled progression to where proteins can be tailored so that they have not only the ability to interact with a unique (extra)cellular component in order to produce a pharmacological effect, but their structure can be altered so as to achieve a selective biological disposition. This ability to control the biological dispersion of proteins is becoming a key element in the design of therapeutic polypeptides and proteins, finding application in modalities intended for treating HIV infections, accessible tumor masses, and diseases of the hematopoietic system, etc. For recombinant site-specific systems to be successfully introduced into the clinic, radically different approaches are required during their development and clinical testing. These relate not only to the pharmacokinetics and receptor availability of the drug, but also its pharmacodynamics, its analytics and its safety pharmacology. This contribution examines how the control of the biological disposition of proteins may be achieved using both site-directed mutagenesis, sequence re-ordering, hybrid protein formation, protein resurfacing, as well as synthetic adduction to polymers.

Chromosome Deletion↗
PubMed1995

RNA-protein interactions directed by the 3' end of human rhinovirus genomic RNA.

The replication of a picornavirus genomic RNA is a template-specific process involving the recognition of viral RNAs as target replication templates for the membrane-bound viral replication initiation complex. The virus-encoded RNA-dependent RNA polymerase, 3Dpol, is a major component of the replication complex; however, when supplied with a primed template, 3Dpol is capable of copying polyadenylated RNAs which are not of viral origin. Therefore, there must be some other molecular mechanism to direct the specific assembly of the replication initiation complex at the 3' end of viral genomic RNAs, presumably involving cis-acting binding determinants within the 3' noncoding region (3' NCR). This report describes the use of an in vitro UV cross-linking assay to identify proteins which interact with the 3' NCR of human rhinovirus 14 RNA. A cellular protein(s) was identified in cytoplasmic extracts from human rhinovirus 14-infected cells which had a marked binding preference for RNAs containing the rhinovirus 3' NCR sequence. This protein(s) showed reduced cross-linking efficiency for a 3' NCR with an engineered deletion. Virus recovered from RNA transfections with in vitro transcribed RNA containing the same 3' NCR deletion demonstrated a defective replication phenotype in vivo. Cross-linking experiments with RNAs containing the poliovirus 3' NCR and cytoplasmic extracts from poliovirus-infected cells produced an RNA-protein complex with indistinguishable electrophoretic properties, suggesting that the appearance of the cellular protein(s) may be a common phenomenon of picornavirus infection. We suggest that the observed cellular protein(s) is sequestered or modified as a result of rhinovirus or poliovirus infection and is utilized in viral RNA replication, perhaps by binding to the 3' NCR as a prerequisite for replication complex assembly at the 3' end of the viral genomic RNA.

Base Sequence↗
PubMed2005

3D morphology of cell cultures: a quantitative approach using micrometer synchrotron light tomography.

Current issues in both tissue engineering and cell biology deal with cell behavior extensively in 3D. Here, we explore synchrotron radiation micro-computed tomography as a tool for morphological characterization of such 3D cellular constructs, providing micrometer resolution in soft and hard tissues. Novel image processing techniques allowed quantification of local and global cell distributions, cell density, adhesive cell culture surface, and scaffold geometry. For proof of concept, we applied this technique to characterize the morphology of two cell cultures of different phenotypes, namely human dermal fibroblasts and mouse calvarial osteoblast-like cells, both seeded on a polymer multifilament yarn. From 3D visualizations in these case studies, we saw that the fibroblasts spanned between the yarn filaments and in this way encapsulated the yarn, whereas the osteoblast-like cells lined the filament surfaces and did not span between them. Differences found in cell distribution as a function of distance to the median yarn axis and the closest filament surface, respectively, quantified these qualitative impressions gained from 3D visualizations. Moreover, the volume-normalized adhesive surface differed by one order of magnitude between the two phenotypes. Our approach allows quantitative correlation of local scaffold geometry and cell morphology. It can be used to investigate the influence of cell phenotype as well as various biochemical agents on tissue engineering constructs and the behavior of cells in culture.

Animals↗
PubMed2004

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases.

The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism. It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes. In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca(2+) homeostasis machinery, with particular emphasis on Ca(2+)-transporting ATPases. Putative Ca(2+)-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca(2+) pumps. High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca(2+) transport and trafficking. The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca(2+),Mn(2+)-ATPases (SPCA). Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump. Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin. Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease.

Animals↗
PubMed2005

Re-engineering redox-sensitive green fluorescent protein for improved response rate.

Redox-sensitive variants of the green fluorescent protein (roGFPs) had previously been developed that allow "real-time" monitoring of the redox status of cellular compartments by fluorescence excitation ratiometry. However, the response time of these probes limits the study of certain rapid oxidative events, such as H2O2 bursts in cell signaling. The substitution of up to three positively charged amino acids adjacent to the introduced disulfide in roGFP1 (variants designated roGFP1-R1 through -R14) substantially improved the response rate. The pseudo first-order rate constants for oxidation by H2O2 and reduction by DTT and redox midpoint potentials were determined. The rate constants approximately doubled with each additional positively charged substitution, to nearly an order of magnitude total. The midpoint potentials are highly correlated with the rate increases, becoming more oxidizing with increasing numbers of positive substitutions. Crystal structures of two variants with opposite disulfide oxidation states have been determined: a 2.2 A resolution structure of oxidized "R7" containing two basic substitutions, and a 1.95 A resolution structure of reduced "R8" with one basic and one acidic substitution. Nonlinear Poisson-Boltzmann (PB) calculations are shown to accurately predict the effects of the substitutions on the rate constants. The effects of the substitutions on dimer formation, relative oxidative midpoint potentials, and oxidation and reduction rates are discussed. roGFPs are demonstrated to constitute an excellent model system for quantitative analysis of factors influencing thiol transfer reactions. roGFP1-R12 is most suitable for use in live cells, due to significantly increased reaction rate and increased pI.

Amino Acid Substitution↗
PubMed1997

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

Animals↗
PubMed2000

A study of injectable tissue-engineered autologous cartilage.

OBJECTIVE: To test the effectiveness of the new techniques of tissue-engineered cartilage. METHODS: Chondrocytes were harvested through type II collagenase digestion from the auricle of New Zealand rabbits. The cells were mixed with alginate to generate chondrocytes/alginate composites with final cellular density of 50 x 10(6) per mL. Calcium chloride was used as the cross-linking agent to gel the aqueous alginate solution. The chondrocytes/alginate composites were injected into the dorsal subcutaneous tissue of New Zealand rabbits through autologous cells grafts. The specimens were observed during cartilage formation at 4, 8, and 12 weeks after injection. RESULTS: Prior to harvesting, chondrocytes/alginate composites were easily visualized under the dorsal skin of animals. The appearance of experimental specimens was similar to that of native cartilage in gross morphology. Using a standard hematoxylin and eosin stain, the histologic features of all experimental specimens demonstrated new cartilage formation. With a Masson's trichrome and safranin O stain, the presence of collagen and glycosaminoglycan (GAG) was observed at 8 and 12 weeks. CONCLUSION: This study demonstrated that polymerization of alginate hydrogel can be controlled to allow injection of chondrocytes that produce new autologous cartilage at subcutaneous dorsal site of rabbits. Injectable tissue-engineered autologous cartilage is promising for potential use in oral and maxillofacial surgery.

Alginates↗
PubMed2004

Biological and biophysical principles in extracorporal bone tissue engineering. Part II.

The aim of this review is to characterise the biological and biophysical background of in vitro bone tissue engineering. The paper focuses on basic principles in extracorporal engineering of bone-like tissues, considering parameters such as scaffold design, tissue construction, bioreactors, and cell stimulation in vivo and in vitro. Scaffolds have a key function concerning cellular invasion and bone formation. The intra-architectural scaffold geometry, as well as the scaffold material, play an important role in the process of bone regeneration. Various types of bioreactors have been tested for their utility in bone substitute fabrication that is clinically effective and reproducible. Sophisticated bioreactor systems are those that mimic the three-dimensional morphology and the mechanical situation of bones. Mechanical stimulation as well as other biophysical stimuli appear to be critical factors for proliferation and differentiation of bone cells and for bone mineral and structure formation. Furthermore an enhancement of bone regeneration by application of chemical stimulation factors is discussed.

Animals↗
PubMed2002

Genomic and proteomic perspectives in cell culture engineering.

In the last few years, the number of biologics produced by mammalian cells have been steadily increasing. The advances in cell culture engineering science have contributed significantly to this increase. A common path of product and process development has emerged in the last decade and the host cell lines frequently used have converged to only a few. Selection of cell clones, their adaptation to a desired growth environment, and improving their productivity has been key to developing a new process. However, the fundamental understanding of changes during the selection and adaptation process is still lacking. Some cells may undergo irreversible alteration at the genome level, some may exhibit changes in their gene expression pattern, while others may incur neither genetic reconstruction nor gene expression changes, but only modulation of various fluxes by changing nutrient/metabolite concentrations and enzyme activities. It is likely that the selection of cell clones and their adaptation to various culture conditions may involve alterations not only in cellular machinery directly related to the selected marker or adapted behavior, but also those which may or may not be essential for selection or adaptation. The genomic and proteomic research tools enable one to globally survey the alterations at mRNA and protein levels and to unveil their regulation. Undoubtedly, a better understanding of these cellular processes at the molecular level will lead to a better strategy for 'designing' producing cells. Herein the genomic and proteomic tools are briefly reviewed and their impact on cell culture engineering is discussed.

Animals↗