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Efforts to manipulate living organisms have raised the question of whether engineering principles of hierarchy, abstraction and design can be applied to biological systems. Here, we consider the practical challenges to controlling living organisms that must be surmounted, or at least managed, if synthetic biology and cellular bioengineering are to be productive.
Bordetella pertussis is the causative agent of whooping cough. The cellular pertussis vaccine introduced in the forties is highly effective and is widely used, but its reactogenicity has led to public concern regarding its safety. The attempts to reduce the side effects associated with pertussis immunization have led to the preparation of acellular B. pertussis products: one composed of detoxified pertussis toxin (PT) and filamentous haemagglutinin (FHA), another one composed only of detoxified PT and a third vaccine composed of detoxified PT, FHA and serotypes 1, 3 of fimbriae. In our laboratories we have approached the study of pertussis toxin, the molecule present in all the proposed acellular pertussis vaccines and one of the main virulence factors of B. Pertussis, with the aim of producing new acellular pertussis vaccines by using recombinant DNA techniques.
Cell culture in a biomimetic environment is known to improve the mechanical endurance of tissue-engineered cardiovascular components. Our goal was to generate a bioreactor that can reproduce a wide range of pulsatile flows with a completely physiological pressure profile. The morphology and biochemical properties of tissue-engineered products were also studied to test the usefulness of this novel bioreactor. The combination of an outflow valve, compliance chamber, and resistant clamps together with a balloon pumping system was able to successfully reproduce both physiological systolic and diastolic pressures. The compliance chamber was especially effective in transforming the original peaky pressure waveform into a physiological pressure profile. The tissues, cultured under a physiological pressure waveform with pulsatile flow, presented widely distributed cells in close contact with each other. They also showed significantly higher cell numbers, total protein content, and proteoglycan-glycosaminoglycan content than cultured tissues under a peaky pressure wave or under static conditions. This new bioreactor system is suitable for evaluating a favorable environment for tissue-engineered cardiovascular components.
Despite enormous advances made in recent years, there remain fundamental and fascinating unanswered questions in immunology. They would include the mechanism of tolerance, the biological significance of the major histocompatibility complex (MHC) restriction, generation of diversity in T cells, control of V region and isotype selection in B cells, how immune response genes work and how important their function is in man, among others. With the advent of monoclonal antibodies and recombinant DNA technologies, together with greater understanding of immunological interactions within the network and of the intracellular networks between membrane-associated receptors and cell function, a variety of immunological strategies are available for modulating immune responses in man. Some nonspecific strategies include immunomodulatory lymphokines, receptor modulation, and development of agonists and antagonists for important receptors. In addition, there is great potential for immunologically specific strategies for intervention, including the use of idiotypes and anti-idiotypes and genetic engineering of antibodies. Ultimately, there is the possibility of predicting antigenic epitopes likely to engage the cellular and humoral arms of the immune response on the basis of protein or DNA sequences. The potential of exploring these developments for new diagnostic tests and vaccines is emphasized here, as is the unpredictable importance of seemingly nonrelevant fundamental science in producing many of the tools for intervention, now and in the future.
Peroxisome proliferator-activated receptor-gamma (PPARgamma) is expressed in the intestinal epithelium, yet little is known about the physiological role of PPARgamma in the small bowel or the effects of PPARgamma on small intestinal epithelial cells. The present studies investigate cellular and genomic effects of PPARgamma in nontransformed rat intestinal epithelial cells (RIE). These cells were engineered to express mouse PPARgamma1, and thereby to model the molecular phenotype that obtains upon induction of PPARgamma at the crypt/villus junction in the small intestine. In these studies, we have used a novel third generation thiazolidinedione derivative, RS5444, which activates PPARgamma with an EC50 about 1/50th that of rosiglitazone and has no effect on RIE cells that do not express PPARgamma. We used Affymetrix oligonucleotide microarrays to identify potential PPARgamma-regulated processes in RIE cells, including lipid metabolism, cell proliferation and differentiation, remodeling of the extracellular matrix, cell morphology, cell-cell adhesion, and motility. The genomic profile reflects cellular events that occur following PPARgamma activation: RS5444 inhibited culture growth and caused irreversible G1 arrest, but did not induce apoptosis. In addition, RS5444 caused dramatic changes in cellular morphology which were associated with increased motility and diminished cellular adherence, but no increase in the ability of such cells to digest and invade Matrigel. Inhibition of proliferation, cell cycle arrest, increased motility, and altered adherence are aspects of the differentiated phenotype of villus epithelial cells, which withdraw from the cell cycle at the crypt/villus interface, migrate to the villus tips, and are subsequently shed by loss of contact with the epithelium and the underlying extracellular matrix. Our results are consistent with the hypothesis that PPARgamma regulates critical aspects of differentiation in the small intestinal epithelium. Many nuclear receptors regulate differentiation. However, our results point to novel effects of PPARgamma on cell-cell and cell-matrix interactions, which are not typical of other nuclear receptors.
Three proteins are functionally interlinked in the targeting of protein phosphorylation catalyzed by the C-subunit of PKA: PKA itself, AKAPs and NMT. Furthermore, in a variety of biological contexts, mechanisms exist whereby PKA and PKC are able to modulate the activity of one another. We have investigated the expression and subcellular distribution of these proteins in two models of mammary cell proliferation and differentiation--the normal rat mammary gland during pregnancy and lactation and human breast tissue before and after malignant transformation. Modulation of PKA does not acutely affect activity or sub-cellular distribution of PKC in mammary acini, nor does modulation of PKC acutely affect PKA activity or subcellular distribution. Therefore, the co-ordinate expression of these two protein kinases in normal and cancerous mammary epithelial cells and the greater basal activation level of them both accompanying increased mitogenic activity, which we have reported, does not result from short-term cross-talk between them. Although basal and total levels of PKA diminish in rodent mammary epithelial cells during the transition from proliferative to secretory functional mode, the level of expression of AKAPs increases. The expression of two apparently mammary-specific and mostly membrane-associated AKAPs is tightly linked to the onset and maintenance of differentiated function in rat mammary tissue. Paradoxically, the probable analogues of these two AKAPs in human mammary tissue are hyperexpressed when normal epithelial cells transform to a cancer phenotype--conventionally regarded as a process involving a degree of dedifferentiation. Mammary AKAP hyperexpression in breast cancers is accompanied by increases in the levels of total and basal PKA. One mechanism whereby NMT is targeted to membranes, via interaction with ribosomal proteins, has recently been elucidated. Our data support the contention that the localization of NMT is an important variable in the regulation of cellular proliferation, but they do not characterize the mechanisms whereby the differential targeting of NMT is achieved. As yet we lack a full tool-kit with which to examine NMT either to draw firm conclusions regarding the identity of particular isoforms found in particular sub-cellular locations or to define the relationships between these different molecular variants. However, it is technically possible to transfect cells with inducible NMT expression constructs engineered in such a way that the recombinant, catalytically competent, NMT that they encode is targeted either to membranes or to cytosol: an exploration of the effects of such transfections on cellular proliferation would afford a critical test of the mechanistic involvement of NMT in the control of mitogenesis.
The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.
G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.
Abeta peptides, which are believed to be at the origin of Alzheimer's disease (AD), are produced through the sequential processing of the transmembrane amyloid precursor protein (APP) by the beta- and gamma-secretase. The identification of small molecules that penetrate the brain and inhibit these secretases is of great therapeutic potential. Here, we describe a cellular selection system in yeast for the identification of inhibitors of the human beta-secretase BACE-1. Similar to the natural situation in mammalian cells, BACE-1 and its substrate APP are bound to membranes in secretory pathway compartments. Yeast cells expressing these human proteins have been engineered so as to grow under selective conditions only upon reduction of BACE-1 activity, thus allowing identification of compounds that, in addition to inhibiting BACE-1, must permeate cellular membranes and present no cytotoxic effects. Our results show that gradual reduction of BACE-1 expression in the engineered yeast strain resulted in gradual increase of cell growth rate. Moreover, two validated BACE-1 inhibitors, which have IC50 values between 7 and 8 microM in mammalian cell assays, stimulated yeast growth in a concentration-dependent manner. This effect was specific for BACE-1 since these compounds had no effect on yeast cells expressing a different secretase cleaving the APP substrate at the alpha-site. The target-specific cellular assay presented here is applicable in high-throughput screens for selecting inhibitors of defined secretases acting on natural substrates in a membrane-bound protein configuration.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signal transduction program termed the unfolded protein response (UPR). In mammalian cells, the UPR is signaled in part through dimerization of ER membrane-localized IRE1alpha to activate its protein kinase and endoribonuclease activities. Activated IRE1alpha cleaves XBP1 mRNA at two sites to initiate an unconventional splicing reaction. The 5' and 3' fragments are subsequently joined by an RNA ligase activity, thereby removing a 26-base intron. This splicing reaction creates a translational frameshift to produce a functional XBP1 transcription factor. However, the cellular location and physiological processes required for splicing of XBP1 mRNA are not well characterized. To study these processes, XBP1 mRNAs were engineered in which translation of enhanced green fluorescence protein or luciferase required splicing of the XBP1 intron. Using cell lines that continuously or transiently express these reporter constructs, we show that cytoplasmic unspliced XBP1 mRNA is efficiently spliced by activated IRE1alpha and requires ongoing cellular transcription but not active translation. The XBP1 intron was effectively removed from RNA substrates transcribed from T7 RNA polymerase or delivered directly to the cytoplasm by RNA transfection, thus indicating that the splicing reaction does not require nuclear processing of the RNA substrate. Analysis of nuclear and cytoplasmic RNA fractions demonstrated that XBP1 mRNA splicing occurs in the cytoplasm. Moreover, an artificial F(v)-IRE1alphaDeltaN was engineered that was able to splice XBP1 mRNA upon chemical-induced dimerization. These findings demonstrate that IRE1alpha dimerization is sufficient to activate XBP1 mRNA splicing in the absence of the UPR. We propose that XBP1 mRNA cytoplasmic splicing provides a novel mechanism to rapidly induce translation of a transcription factor in response to a specific stimulus.
Decay-accelerating factor (DAF) has been reported to be a cellular receptor for several enteroviruses. Buffalo green monkey kidney (BGMK) cells expressing human DAF (BGMK-hDAF cells) showed increased susceptibility and sensitivity to several types of enteroviruses compared to wild-type BGMK cells. When 17 frozen positive clinical samples were tested, BGMK cells detected 8 and BGMK-hDAF cells detected 16. Since the CaCo-2 cell line has been documented to support the replication of most enteroviruses, CaCo-2 cells were mixed with BGMK-hDAF cells in order to increase the number of viruses detected. Thirty-four frozen clinical samples that previously had tested positive for enteroviruses were tested, and the following numbers were detected: 33 of 34 by CaCo-2/BGMK-hDAF cells, 29 of 34 by CaCo-2/BGMK cells, 28 of 34 by H292/RD (E-mix A) and A-549/BGMK (E-mix B) cells, and 26 of 34 by MRC-5 and pRhMK cells.
In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr(-) cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH > anti-LDH > GP3 > CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 > CHO > GP3/anti-LDH > anti-LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time ( approximately 19 h compared with approximately 26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO(3) (-)-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of recombinant proteins.
Engineered tissues can be employed for studies on the fundamental mechanisms of embryology and adult physiology and for investigating the evolution of disease processes. They also provide platforms to evaluate the behavior of new chemical entities in drug development. The recent development of three specific technologies has greatly facilitated the engineering of tissues for in vitro applications: the microfabrication tools that serve to both define the cellular microenvironment and enable parallelization of cell-based assays; synthetic, tunable hydrogels to create three-dimensional microenvironments; and bioreactors to control nutrient transport and fluid shear stress. Furthermore, convergence of these tools is providing investigators with the opportunity to construct and study tissues in vitro with unprecedented levels of sophistication.
Dual ligand treatment of streptavidin(SA)-biotin and fibronectin (Fn) enhances the adhesion of endothelial cells (EC) onto synthetic surfaces and promotes the quiescent phenotype of adherent EC. The current study investigates the effect of the dual ligand on the expression of endothelial genes in static culture and under shear stress (4 h at 10 dynes/cm2). Expression of 23 genes in the classes of signaling, cytoskeleton/ECM, vasoregulation, and shear-responsive were examined. Eight genes (argininosuccinate synthetase, K+ channel, TGFbeta, Mn-SOD, alpha-tubulin, t-PA, COX2, and eNOS) were significantly upregulated by shear stress. Two genes (caveolin-1 and ET-1) were downregulated by shear stress. Three genes (RhoA, elastin, alpha-actinin) were upregulated by the dual ligand treatment in static culture, and four genes (FAK, elastin, COX2, and eNOS) were upregulated when the dual ligand and shear stress were applied simultaneously. Northern blot analyses on FAK, RhoA, elastin, and alpha-actinin revealed similar results. The results suggest (1) the use of SA-biotin to supplement EC adhesion enhances the integrity of the EC cytoskeleton by upregulating the expression of cytoskeleton/ECM genes, and (2) a likely relationship between the expression of cytoskeleton/ECM genes and the downstream events, such as the shear-induced expression of eNOS and COX2 genes. Analyses presented in this study provide insights into the mechanism by which SA-biotin-supplemented EC mediate gene expression.
Surgical treatment of critical-size posttraumatic bone defects is still a challenging problem, even in modern bone and joint surgery. Progress in cellular and molecular biology during the last decade now permits novel approaches in bone engineering. Recent conceptual and technical advances have enabled the use of mitotically expanded, bone-derived cells as a therapeutic approach for tissue repair. Using three different tissue carrier systems, we successfully cultivated human osteoblasts in a newly developed perfusion chamber. We studied cell proliferation and the expression of osteocalcin, osteopontin, bone morphogenetic protein-2A, alkaline phosphatase, and vascular endothelial growth factor as parameters for osteoblast function and viability. Adherence of highly enriched human osteoblasts had already started after 1 h and resulted in completely overgrown human bone pieces after 10 days. Expression analysis of bone-specific alkaline phosphatase indicated differentiating osteoblasts, whereas the high mRNA expression of osteocalcin and osteopontin revealed terminally differentiated osteoblasts and the process of mineralization. Additionally, gene expression was significantly higher when demineralized bone was used as biomatrix, compared to autoclaved bone and hydroxyapatite ceramics. We conclude that with our newly developed perfusion culture system, vital autogenous bone implants of clinically applicable size can be generated within 17 days in order to manage critical-size bone defects.
Nuclear transport factor 2 (NTF2) mediates nuclear import of RanGDP, a central component of many nuclear trafficking pathways. NTF2 is a homodimer and each chain has independent binding sites for RanGDP and nuclear pore proteins (nucleoporins) that contain FxFG sequence repeats. We show here that the monomer-dimer dissociation constant for NTF2 obtained by sedimentation equilibrium ultracentrifugation is in the micromolar range, indicating that a substantial proportion of cellular NTF2 may be monomeric. To investigate the functional significance of NTF2 dimerization, we engineered a series of point mutations at the dimerization interface and one of these (M118E) remained monomeric below concentrations of 150 microM. CD spectra and X-ray crystallography showed that M118E-NTF2 preserved the wild-type NTF2 fold, although its thermal stability was 20 deg. C lower than that of the wild-type. M118E-NTF2 bound both RanGDP and FxFG nucleoporins less strongly, suggesting that dissociation of the NTF2 dimer could facilitate RanGDP release and thus nucleotide exchange after it had been transported into the nucleus. Moreover, colloidal gold coated with M118E-NTF2 showed reduced binding to Xenopus oocyte nuclear pores. Overall, our results indicate that dimer formation is important for NTF2 function and give insight into the formation of heterodimers by mRNA export factors such as TAP1 and NXT1 that contain NTF2-homology domains.
P53 is an attractive target immunotherapy because it is overexpressed in up to one half of all malignancies, and its overexpression often correlates with a worsened prognosis. We wanted to determine the feasibility of targeting wild-type epitopes p53 on human tumor cells. HLA A2.1 transgenic mice were immunized with the immunodominant wild-type p53 peptide epitopes, p53(149-157) and p53(264-272), along with a pan-DR helper epitope peptide in incomplete Freund's adjuvant (IFA). Twelve days later, splenocytes were harvested and stimulated with syngeneic blast cells that had been acid-treated to remove endogenous peptide and p53 peptide-pulsed. The responding cells were subsequently restimulated weekly with acid washed, peptide-pulsed Jurkat cells transfected with HLA A2.1. Peptide specific activity was tested in a chromium release assay. The resulting cytotoxic T cells (CTL) were cloned by limiting dilution. Peptide specific CTL were generated against both p53(149-157) and p53(264-272. Only p53(149-157) specific CTL were able to recognize and lyse cells that overexpressed endogenous p53. CTL clones derived from the p53(149-157) cell line demonstrated high affinity and specificity for p53(149-157) when presented by HLA A2.1+ cells. The p53(149-157) specific CTL were tested for specificity against a variety of cultured human cell lines. The CTL clones only lysed cells that overexpressed p53 in the context of HLA A2.1 and did not lyse cells with normal p53 expression or cells that lacked HLA A2.1 expression. This study demonstrates the possibility of targeting tumors, which overexpress p53, and raises the possibility transferring the high affinity, p53 specific T cell receptors from the murine CTL to human T cells.
Sensitivity to pharmacological challenges has been reported in patients with panic disorder. We have previously validated transgenic mice overexpressing the neurotrophin-3 (NT-3) receptor, TrkC (TgNTRK3), as an engineered murine model of panic disorder. We could determine that TgNTRK3 mice presented increased cellularity in brain regions, such as the locus ceruleus, that are important neural substrates for the expression of anxiety in severe anxiety states. Here, we investigated the sensitivity to induce anxiety and panic-related symptoms by sodium lactate and the effects of various drugs (the alpha2-adrenoceptor antagonist, yohimbine and the adenosine antagonist, caffeine), in TgNTRK3 mice. We found enhanced panicogenic sensitivity to sodium lactate and an increased intensity and a differential pattern of Fos expression after the administration of yohimbine or caffeine in TgNTRK3. Our findings validate the relevance of the NT-3/TrkC system to pathological anxiety and raise the possibility that a specific set of fear-related pathways involved in the processing of anxiety-related information may be differentially activated in panic disorder.