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Gradual soil water depletion results in reversible changes of gene expression, protein profiles, ecophysiology, and growth performance in Populus euphratica, a poplar growing in arid regions.

The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated.

Climate↗

Immunological properties of tumor cells genetically modified to secrete interleukin-2.

The immunological properties of interleukin-2 (IL-2) gene-transduced tumor cells were investigated in mice. A murine ovarian cancer cell line, OVHM, was retrovirally transduced with the human IL-2 gene (OVHM/IL-2) and the neomycin resistance gene (OVHM/Neo). OVHM/IL-2 cells continuously secreted IL-2 detected by ELISA using an antibody specific for human IL-2, and by a bioassay using an IL-2-reactive cell line (CTLL-2). When OVHM cells were inoculated subcutaneously into syngeneic B6C3F1 mice, OVHM/IL-2 cells but not parental (OVHM/P) or OVHM/Neo cells were regressed even though their rate of in vitro growth was comparable. This was not observed in nude mice, indicating the involvement of T lymphocytes in the regression of OVHM/IL-2 cells. The survival of mice inoculated intraperitoneally with OVHM/IL-2 cells was prolonged compared with those inoculated with OVHM/P cells. In irradiation experiments, IL-2 secretion by irradiated OVHM/IL-2 cells was retained for at least 5 days, although in vitro growth and in vivo tumorigenicity of irradiated OVHM/IL-2 cells were completely diminished. When mice were challenged with viable OVHM/P cells, survival of mice previously immunized with irradiated OVHM/IL-2 cells was prolonged compared to those immunized with irradiated OVHM/P cells, indicating a vaccine property of irradiated OVHM/IL-2 cells. Moreover, survival of mice with established ascites was improved upon injection of irradiated OVHM/IL-2 cells, indicating a therapeutic potential of OVHM/IL-2 cells. Tumor cells genetically engineered to secrete IL-2 may therefore be promising candidates for tumor vaccines and may provide a new mode of cancer immunotherapy.

Animals↗

[Expression of human bone morphogenetic protein 7 mRNA after the gene transfection in rabbit bone marrow-derived mesenchymal stem cells].

OBJECTIVE: To detect the expression of human bone morphogenetic protein 7 (hBMP-7) mRNA in rabbit bone marrow-derived mesenchymal stem cells with hBMP-7 gene transfection mediated by retroviral vector. METHODS: Retroviral vector for hBMP-7 gene was constructed that was transferred into the packaging cell PT67 mediated by liposome. hBMP-7 gene-positive cell clones were selected with G418 (600 ng/ml) and amplified to obtain the retroviral supernatant containing the target genes that were subsequently used to transfect rabbit bone marrow-derived mesenchymal stem cells (MSCs). HBMP-7 mRNA expression in the MSCs was examined by way of in situ hybridization and reverse transcriptase-polymerase chain reaction. RESULTS: hBMP-7 retroviral vector was successfully constructed and transferred into PT67 cells with resistance to G418, and after transfection with the recombinant retrovirus, transcription and expression of hBMP-7 mRNA were detected in MSCs. CONCLUSION: Rabbit bone marrow stem cells transfected with hBMP gene via retroviral vector can secrete the correspondent protein, indicating the possibility of enhancing the osteogenic capacity of MSCs in the study of bone tissue engineering.

3T3 Cells↗

Synthesis of reassortant infectious bursal disease virus in chickens injected directly with infectious clones from different virus strains.

The infectious bursal disease virus (IBDV), a member of the Birnaviridae family, containing a bisegmented double-stranded RNA genome, encodes four structural viral proteins, VP1, VP2, VP3, and VP4, as well as a non-structural protein, VP5. In the present paper, the segment A from two IBDV strains, field isolate ZJ2000 and attenuated strain HZ2, were inserted into one NaeI site by site-directed silent mutagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cytomegalovirus (hCMV) immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000 and pCI-AKHZ2, respectively. Each of the two recombinants was combined with another recombinant pCI plasmid containing the marked segment B of strain HZ2 (pCI-mB), and injected intramuscularly into non-immunized chickens. Two chimeric IBDV strains were recovered from the chickens. Two out of eight chickens in each of two groups showed the bursal histopathological change. The reassortant virus derived from pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low virulence. We have developed a novel virus reverse genetic approach for the study of IBDV. The results also form the basis for investigating the role of VP1 in viral replication and pathogenecity.

Animals↗

Cloning and expression of the cDNA for canine tumor necrosis factor-alpha in E. coli.

We have utilized the reverse transcription polymerase chain reaction (RT-PCR) to clone the protein coding region of canine tumor necrosis factor (TNF-alpha) cDNA. The gene displays 90% sequence homology to the corresponding human TNF-alpha cDNA. The predicted initial translation product is 233 amino acids and shows 88% homology to the human counterpart, and 92% homology with the human putative mature TNF-alpha protein. The canine TNF-alpha clone was used to engineer bacteria to express large amounts of the mature form of recombinant protein. A monoclonal antibody against human TNF-alpha cross-reacted with canine rTNF-alpha using Western blot and ELISA analysis. The purified canine rTNF-alpha had a cytotoxic effect on WEHI 164 clone 13 cells as well as increasing the cell surface expression of major histocompatibility class II antigens on canine kidney cortical cell line (MDCK) in vitro. The availability of canine rTNF-alpha will allow further studies on its role in immunoregulatory mechanisms in the canine transplantation model, both by itself and in conjunction with the already available canine specific recombinant interferon-gamma.

Amino Acid Sequence↗

Phytolectins: natural molecules with immense biotechnological potential.

Lectins are structurally diverse, carbohydrate binding proteins that bind reversibly to specific mono- or oligosaccharides. Their abundance in the plant kingdom suggest that they have diverse roles to perform. They serve as recognition factor between symbiotic nitrogen fixing bacteria and host plants, as a deterrent to phytopathogens like fungi, insects, and animals, as storage protein and as an aid in sexual reproduction in Chlamydomonas, amongst others. The possible application of lectins as a factor in increasing soil fertility and as a biopesticide by genetically engineered organisms is yet to be fully explored by the biotechnologists. However, they are being used by the biomedical scientists and biochemists in blood typing and stimulation of cells for chromosome analysis and gene mapping, in cell separation, identification of complex glycoproteins and typing of bacteria. Cell targeting by lectins in cancer therapy is still in its infancy. This review gives an insight into the potential of these wonder biomolecules in agriculture, biochemistry, cell biology and medicine for the benefit of mankind.

Biotechnology↗

Genetically engineered binding proteins as biosensors for fermentation and cell culture.

The signal-transduction properties and the potential applications of two engineered binding proteins from E. coli were extensively studied. Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP). Both proteins respond to their ligands in the micromolar range. The proteins can be stored at 4 degrees C for at least 5 months. Apparent binding constant, protein concentration, and fluorophore are three major factors that affect the biosensor's responsive ranges. The binding of the ligand is quick and reversible in solution, but the unfavorable dissociation equilibrium and mass-transfer resistance for encapsulated proteins can delay the response to several minutes and the recovery to hours. Simulated results show that using dialysis tubing with a diameter of 1 mm or less is possible to reduce the recovery time to less than 30 minutes. The potential applications of GBP were studied in yeast fermentation and E. coli fermentations in three different scales: 150 mL, 5 mL, and 100 microL. The results were compared with an YSI 2700 Chemistry Analyzer. Although the latter could not give reliable results for the E. coli fermentations as the glucose concentration in LB medium is close to its lower detection limit, the glucose biosensor presented here was successfully applied to each situation. Glutamine-binding protein was tested in cell cultures of two different scales (100 mL and 100 microL) and the results were also compared with those obtained with YSI. Both QBP and YSI gave good results for the 100-mL cell culture, but the relatively large sample volume requirement of YSI (at least 5 microL) prevented it from being used in the 100-microL cell culture. Because of their small sample volume requirements (less than 1 microL) and high sensitivity, the assays described here might find wide applications in high-throughput bioprocessing.

Bioreactors↗

Gas-inducible product gene expression in bioreactors.

Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals.

Acetaldehyde↗

Electrospray ionization mass spectrometry of recombinantly engineered antibody fragments.

Mass spectrometry has been used to confirm the correct protein expression of Fab and F(ab')2 fragments of the humanized anti-p185HER2 antibody huMAb4D5. These data demonstrate that electrospray ionization mass spectrometry can be used routinely to measure the masses of purified proteins as large as 100 kDa, with an accuracy of < or = 0.02%. This level of accuracy is helpful in demonstrating the faithful translation and proper posttranslational modification of these proteins. Accurate and reliable mass assignments by electrospray ionization mass spectrometry are achieved by calibrating with protein mass standards, optimizing resolution, and using methods to improve sample purity. On-line reversed-phase high-performance liquid chromatography/mass spectrometry has been employed to improve sample purity and thereby increase sensitivity. Disulfide reduction to yield the component subunits provides additional confirmation of the correct amino acid sequence with greater absolute mass accuracy. A volatile reducing agent such as tributylphosphine provides a convenient method to generate subunits for mass measurement while requiring little or no further sample purification.

Humans↗

Cell replacement therapy for type 1 diabetes.

Replacement of the insulin-producing pancreatic islet beta cells represents the ultimate treatment for type 1 diabetes. Recent advances in islet transplantation underscore the urgent need for developing alternatives to human tissue donors, which are scarce. Two possible approaches are the expansion of differentiated beta cells by reversible immortalization and the generation of insulin-producing cells from embryonic or adult stem cells. It is possible that new insights into endocrine pancreas development will ultimately lead to manipulation of progenitor-cell fate towards the beta-cell phenotype of insulin production, storage and regulated secretion. Both allogeneic and autologous surrogate beta cells are likely to require protection from recurring autoimmunity. This protection might take the form of tolerization, cell encapsulation, or cell engineering with immunoprotective genes. If successful, these approaches could lead to widespread cell replacement therapy for type 1 diabetes.

Adult↗

Experimental and theoretical study of processes leading to steady-state sound in annular thermoacoustic engines.

This paper gives a simplified analytical description of spontaneous generation and finite amplitude saturation of sound in annular thermoacoustic engines, and also provides comparison with experiments. The model includes the precise description of thermoacoustic amplification of sound (induced by interaction between an heterogeneously heated stack of solid plates and resonant gas oscillations), which accounts for the details of the temperature distribution in the whole thermoacoustic device (i.e., which does not only account for the mean temperature gradient along the stack). The saturation of the acoustic wave amplitude is described by taking into account both the reverse influence of high amplitude acoustic field on temperature field, and the dissipation of acoustic energy due to higher harmonics generation and minor losses (vortex generation). From the comparison between simulation results and experiments, it is demonstrated that the dynamical behavior observed in our experimental device is predominantly controlled by the effects of acoustic streaming and acoustically enhanced thermal conductivity tending not only to reduce the externally imposed temperature gradient along the stack, but also to change the shape of the temperature field.

Journal Article↗

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.

3T3 Cells↗

Current and emerging therapies for the lysosomal storage disorders.

Targeted treatments for the lysosomal storage disorders (LSDs), in the form of enzyme replacement and/or substrate depletion, have been shown to be relatively safe and effective in reversing core disease features in selected clinical subtypes (including Gaucher disease types I and III, Fabry disease and the Hurler-Scheie syndrome). These approaches have expanded the therapeutic options available to patients with rare genetic disorders, beyond palliative measures (such as liver or kidney transplantation for end-organ failure) and cellular replacement through bone marrow transplantation. Present efforts are focused on the development of novel strategies, including chaperone-mediated enzyme enhancement and genetically engineered stem cell therapy. In the coming decades, a broadening therapeutic horizon for patients with inborn errors of metabolism is anticipated, and the growing experience in the management of patients with LSDs will serve as an instructive model. Among the many challenges will be determination of the extent to which these therapies have modified the course of disease beyond merely extending the age of survival, but also enabling a meaningful patient quality of life, and the minimisation of current resource use. The projected lifetime acquisition costs of newly introduced therapeutic options also raises several issues, related to equitable access and the large opportunity costs for other therapeutic areas, that will need to be addressed by healthcare policy makers and third-party payers.

Animals↗

Growth of mesenchymal stem cells on electrospun type I collagen nanofibers.

We reconstituted type I collagen nanofibers prepared by electrospin technology and examined the morphology, growth, adhesion, cell motility, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) on three nano-sized diameters (50-200, 200-500, and 500-1,000 nm). Results from scanning electron microscopy showed that cells on the nanofibers had a more polygonal and flattened cell morphology. MTS (3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-methoxyphenyl]-2-[4-sul-fophenyl]-2H-tetrazolium compound) assay demonstrated that the MSCs grown on 500-1,000-nm nanofibers had significantly higher cell viability than the tissue culture polystyrene control. A decreased amount of focal adhesion formation was apparent in which quantifiable staining area of the cytoplasmic protein vinculin for the 200-500-nm nanofibers was 39% less compared with control, whereas the area of quantifiable vinculin staining was 45% less for both the 200-500-nm and 500-1,000-nm nanofibers. The distances of cell migration were quantified on green fluorescent protein-nucleofected cells and was 56.7%, 37.3%, and 46.3% for 50-200, 200-500, and 500-1,000 nm, respectively, compared with those on the control. Alkaline phosphatase activity demonstrated no differences after 12 days of osteogenic differentiation, and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed comparable osteogenic gene expression of osteocalcin, osteonectin, and ostepontin between cells differentiated on polystyrene and nanofiber surfaces. Moreover, single-cell RT-PCR of type I collagen gene expression demonstrated higher expression on cells seeded on the nanofibers. Therefore, type I collagen nanofibers support the growth of MSCs without compromising their osteogenic differentiation capability and can be used as a scaffold for bone tissue engineering to facilitate intramembranous bone formation. Further efforts are necessary to enhance their biomimetic properties.

Alkaline Phosphatase↗

Can HIV be Cured? Mechanisms of HIV persistence and strategies to combat it.

Stable remission is the ultimate goal of HIV therapy. A review of recent studies on the ability of HIV to persist despite highly active antiretroviral therapy (HAART) and immune stimulation suggests that achieving this goal will require four developments in basic and clinical science. First, more effective antiretroviral therapies, targeted at proteins other than reverse transcriptase and protease, in order to eliminate the cryptic replication that continues despite best available HAART. Second, agents that activate latent HIV gene expression in quiescent CD4 memory T cells, thereby exposing this viral reservoir to therapeutic intervention by a "shock and kill" strategy. Third, molecules such as immunotoxins that specifically recognize HIV-encoded membrane proteins and thereby potentiate the destruction of infected cells. Fourth, and still most distant, novel approaches such as genetically engineered cytotoxic T lymphocytes or anti-HIV microbes to suppress rekindling of infection by residual virus sequestered in anatomical and cellular reservoirs. Although each of these steps will be difficult to achieve, the many benefits of a cure for HIV make this a worthwhile pursuit.

Anti-HIV Agents↗

Stabilization of short collagen-like triple helices by protein engineering.

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.

Amino Acid Sequence↗

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine.

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.

Animals↗

Modulation of chondrocytic properties of fat-derived mesenchymal cells in co-cultures with nucleus pulposus.

Human subcutaneous fat-derived mesenchymal cells recently have been shown to have the potential to differentiate in vitro into a variety of cell types, including adipocytes, osteoblasts, chondrocytes, and myoblasts. This effect suggests that fat tissue may serve as an abundant and easily acquired source of multipotent cells for tissue engineering. The multipotential characteristics of fat-derived mesenchymal cells from the inguinial fat pad of rabbit have not been clearly defined. In this study we have isolated a population of mesenchymal cells from inguinal fat from adult New Zealand white rabbits. The cells that were maintained under various differentiation conditions were shown to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes; this differentiation was demonstrated using gene expression for tissue-specific proteins. We also co-cultured the cells with intervertebral disk tissue from the nucleus pulpous or from the annulus fibrosus. The fat-derived cells co-cultured with nucleus pulposus showed an increase in expression of type II collagen and aggrecan genes, compared with cells in alginate alone and cells co-cultured with annulus fibrosus. The data suggest that the fat-derived mesenchymal cells responded to soluble mediators from the disk. Future studies on intervertebral disk reconstruction could be based on our findings with fat-derived multipotential cells from the inguinal region of the rabbit that were co-cultured with disk tissue and may prove useful in tissue engineering strategies.

Adipose Tissue↗