Basic science and clinical application of stem cells in veterinary medicine.

Stem cells play an important role in veterinary medicine in different ways. Currently several stem cell therapies for animal patients are being developed and some, like the treatment of equine tendinopathies with mesenchymal stem cells (MSCs), have already successfully entered the market. Moreover, animal models are widely used to study the properties and potential of stem cells for possible future applications in human medicine. Therefore, in the young and emerging field of stem cell research, human and veterinary medicine are intrinsically tied to one another. Many of the pioneering innovations in the field of stem cell research are achieved by cooperating teams of human and veterinary medical scientists.Embryonic stem (ES) cell research, for instance, is mainly performed in animals. Key feature of ES cells is their potential to contribute to any tissue type of the body (Reed and Johnson, J Cell Physiol 215:329-336, 2008). ES cells are capable of self-renewal and thus have the inherent potential for exceptionally prolonged culture (up to 1-2 years). So far, ES cells have been recovered and maintained from non-human primate, mouse (Fortier, Vet Surg 34:415-423, 2005) and horse blastocysts (Guest and Allen, Stem Cells Dev 16:789-796, 2007). In addition, bovine ES cells have been grown in primary culture and there are several reports of ES cells derived from mink, rat, rabbit, chicken and pigs (Fortier, Vet Surg 34:415-423, 2005). However, clinical applications of ES cells are not possible yet, due to their in vivo teratogenic degeneration. The potential to form a teratoma consisting of tissues from all three germ lines even serves as a definitive in vivo test for ES cells.Stem cells obtained from any postnatal organism are defined as adult stem cells. Adult haematopoietic and MSCs, which can easily be recovered from extra embryonic or adult tissues, possess a more limited plasticity than their embryonic counterparts (Reed and Johnson, J Cell Physiol 215:329-336, 2008). It is believed that these stem cells serve as cell source to maintain tissue and organ mass during normal cell turnover in adult individuals. Therefore, the focus of attention in veterinary science is currently drawn to adult stem cells and their potential in regenerative medicine. Also experience gained from the treatment of animal patients provides valuable information for human medicine and serves as precursor to future stem cell use in human medicine.Compared to human medicine, haematopoietic stem cells only play a minor role in veterinary medicine because medical conditions requiring myeloablative chemotherapy followed by haematopoietic stem cell induced recovery of the immune system are relatively rare and usually not being treated for monetary as well as animal welfare reasons.In contrast, regenerative medicine utilising MSCs for the treatment of acute injuries as well as chronic disorders is gradually turning into clinical routine. Therefore, MSCs from either extra embryonic or adult tissues are in the focus of attention in veterinary medicine and research. Hence the purpose of this chapter is to offer an overview on basic science and clinical application of MSCs in veterinary medicine.

Divergent transcriptional responses to independent genetic causes of cardiac hypertrophy.

To define molecular mechanisms of cardiac hypertrophy, genes whose expression was perturbed by any of four different transgenic mouse hypertrophy models [protein kinase C-epsilon activation peptide (PsiepsilonRACK), calsequestrin (CSQ), calcineurin (CN), and Galpha(q)] were compared by DNA microarray analyses using the approximately 8,800 genes present on the Incyte mouse GEM1. The total numbers of regulated genes (tens to hundreds) correlated with phenotypic severity of the model (Galpha(q) > CN > CSQ > PsiepsilonRACK), but demonstrated that no single gene was consistently upregulated. Of the three models exhibiting pathological hypertrophy, only atrial natriuretic peptide was consistently upregulated, suggesting that transcriptional alterations are highly specific to individual genetic causes of hypertrophy. However, hierarchical-tree and K-means clustering analyses revealed that subsets of the upregulated genes did exhibit coordinate regulatory patterns that were unique or overlapping across the different hypertrophy models. One striking set consisted of apoptotic genes uniquely regulated in the apoptosis-prone Galpha(q) model. Thus, rather than identifying a single common hypertrophic cardiomyopathy gene program, these data suggest that extensive groups of genes may be useful for the prediction of specific underlying genetic determinants and condition-specific therapeutic approaches.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo.

The Ca(2+)-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. DeltaCain and DeltaA-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of DeltaCain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. DeltaCain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and slow myosin heavy-chain expression.

The differentiation and maturation of skeletal muscle cells into functional fibers is coordinated largely by inductive signals which act through discrete intracellular signal transduction pathways. Recently, the calcium-activated phosphatase calcineurin (PP2B) and the family of transcription factors known as NFAT have been implicated in the regulation of myocyte hypertrophy and fiber type specificity. Here we present an analysis of the intracellular mechanisms which underlie myocyte differentiation and fiber type specificity due to an insulinlike growth factor 1 (IGF-1)-calcineurin-NFAT signal transduction pathway. We demonstrate that calcineurin enzymatic activity is transiently increased during the initiation of myogenic differentiation in cultured C2C12 cells and that this increase is associated with NFATc3 nuclear translocation. Adenovirus-mediated gene transfer of an activated calcineurin protein (AdCnA) potentiates C2C12 and Sol8 myocyte differentiation, while adenovirus-mediated gene transfer of noncompetitive calcineurin-inhibitory peptides (cain or DeltaAKAP79) attenuates differentiation. AdCnA infection was also sufficient to rescue myocyte differentiation in an IGF-depleted myoblast cell line. Using 10T1/2 cells, we demonstrate that MyoD-directed myogenesis is dramatically enhanced by either calcineurin or NFATc3 cotransfection, while a calcineurin inhibitory peptide (cain) blocks differentiation. Enhanced myogenic differentiation directed by calcineurin, but not NFATc3, preferentially specifies slow myosin heavy-chain expression, while enhanced differentiation through mitogen-activated protein kinase kinase 6 (MKK6) promotes fast myosin heavy-chain expression. These data indicate that a signaling pathway involving IGF-calcineurin-NFATc3 enhances myogenic differentiation whereas calcineurin acts through other factors to promote the slow fiber type program.

Identification of Saccharomyces cerevisiae genes conferring resistance to quinoline ring-containing antimalarial drugs.

To identify genes that can confer resistance to antimalarial drugs in yeast, we transformed the quinidine-sensitive strain CYX247-9A of Saccharomyces cerevisiae with a yeast genomic library and selected for transformants that grow in the presence of elevated levels of antimalarial drugs. Plasmids were rescued from such clones and were analyzed for the presence of individual open reading frames that can confer drug resistance. Using quinidine as the selective drug, we were able to identify three genes that can cause resistance to antimalarial drugs. Overexpression of the yeast genes CIN5 (a member of the family of bZIP transcription factors), STII (a Hsp90 cochaperone), and YOR273c (a member of the major facilitator superfamily of transmembrane transporters) conferred 3.9-, 7.0-, and 4.3-fold resistance to quinidine, respectively, over that of control yeast. Cross-resistance assays determined that STI1 also conferred resistance to mefloquine (3.4-fold), while CIN5 also conferred resistance to mefloquine (9.6-fold) and chloroquine (5.4-fold). Using mefloquine as the selective drug, we determined that overexpression of YBR233w, a member of the hnRNPK family of nuclear RNA binding proteins, conferred resistance to mefloquine (13.5-fold). Expression of the human hnRNPK homolog of YBR233w in S. cerevisiae also conferred mefloquine resistance, suggesting that homologs of the identified resistance genes may perform similar functions in species other than yeast. Our experiments have identified heretofore unknown pathways of resistance to quinoline ring-containing antimalarial drugs in S. cerevisiae.

Cloning and sequence analysis of a novel member of the ATP-binding cassette (ABC) protein gene family from Plasmodium falciparum.

We have employed oligonucleotide primers directed against the Walker A and B ATP-binding consensus motifs in a PCR-approach to clone a novel member of the eukaryotic ABC protein family of genes from Plasmodium falciparum. The novel gene is predicted to encode a 95.5-kDa protein with two ATP-binding folds each containing a Walker A and B consensus motif and an ABC protein signature sequence. The predicted protein is highly hydrophilic and contains numerous phosphorylation consensus sites but does not contain any potential membrane spanning domains. The gene is present on chromosome 11 and is expressed as a 3.3-kb transcript. The closest homologue with known function to the plasmodial gene is the yeast GCN20 gene which is part of the translation initiation pathway in amino acid starved yeast cells. We have therefore tentatively named the gene Plasmodium falciparum GCN20 homologue (pfgcn20). The pfgcn20 encoded Pfgcn20 protein is also highly homologous to a number of ATP-binding subunits of prokaryotic ABC transporters. We speculate that Pfgcn20 may be an example of a eukaryotic ATP-binding cytosolic subunit of a multipeptide ABC transporter.

The pfmdr1 gene of Plasmodium falciparum confers cellular resistance to antimalarial drugs in yeast cells.

The exact role of the pfmdr1 gene in the emergence of drug resistance in the malarial parasite Plasmodium falciparum remains controversial. pfmdr1 is a member of the ATP binding cassette (ABC) superfamily of transporters that includes the mammalian P-glycoprotein family. We have introduced wild-type and mutant variants of the pfmdr1 gene in the yeast Saccharomyces cerevisiae and have analyzed the effect of pfmdr1 expression on cellular resistance to quinoline-containing antimalarial drugs. Yeast transformants expressing either wild-type or a mutant variant of mouse P-glycoprotein were also analyzed. Dose-response studies showed that expression of wild-type pfmdr1 causes cellular resistance to quinine, quinacrine, mefloquine, and halofantrine in yeast cells. Using quinacrine as substrate, we observed that increased resistance to this drug in pfmdr1 transformants was associated with decreased cellular accumulation and a concomitant increase in drug release from preloaded cells. The introduction of amino acid polymorphisms in TM11 of Pgh-1 (pfmdr1 product) associated with drug resistance in certain field isolates of P. falciparum abolished the capacity of this protein to confer drug resistance. Thus, these findings suggest that Pgh-1 may act as a drug transporter in a manner similar to mammalian P-glycoprotein and that sequence variants associated with drug-resistance pfmdr1 alleles behave as loss of function mutations.

Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA.

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat.

The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.

The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.

A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions +23 to +25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position +23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions +31 to +34) and the stem (positions +9 to +12 and +49 to +52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.

Systematic studies on parameters influencing the performance of the polymerase chain reaction.

To obtain a detailed understanding of the various factors influencing the polymerase chain reaction, we optimized parameters such as ion concentration, pH, primer sequence and concentration, hybridization stringency, cycle numbers, etc. Using 3 different plasmids (2 HIV2 clones and pUC18) and several genomic DNA samples from HIV-positive patients as templates, together with 6 sets of primers, the following optimal conditions were found: the DNA should be linearized, the primer concentration should be 0.1-0.2 mumol/l, and the magnesium ion concentration should be less than 2 mmol/l. The pH of the reaction mixture should be 8.5-9.0. Twenty five cycles are sufficient. For fragments greater than 10(3) bases the elongation time should be 5 min. The elongation temperature is not critical and can vary between 50 and 70 degrees C. The hybridization temperature can be used to control the specificity of the polymerase chain reaction and, finally, mismatches at the 3' end of the primer can totally inhibit the amplification.