Biomedical subjects

A Gavalas

Publications and source records attributed to A Gavalas.

13 recordsLinked to original sources

PubMed2001

The recruitment of SOX/OCT complexes and the differential activity of HOXA1 and HOXB1 modulate the Hoxb1 auto-regulatory enhancer function.

Regionally restricted expression patterns of Hox genes in developing embryos rely on auto-, cross-, and para-regulatory transcriptional elements. One example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere of the hindbrain. We previously showed that HOXB1 and PBX1 activate transcription from the b1-ARE by binding to sequences required for the expression of a reporter gene in rhombomere 4 in vivo. We now report that in embryonal carcinoma cells, which retain characteristics of primitive neuroectodermal cells, the b1-ARE displays higher basal and HOX/PBX-induced activities than in other cell backgrounds. We have identified a bipartite-binding site for SOX/OCT heterodimers within the b1-ARE that accounts for its cell context-specific activity and is required for maximal transcriptional activity of HOX/PBX complexes in embryonal carcinoma cells. Furthermore, we found that in an embryonal carcinoma cell background, HOXB1 has a significantly higher transcriptional activity than its paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using analysis in transgenic and HOXA1 mutant mice, we extended these findings on the differential activities of HOXA1 and HOXB1 in vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of the SOX/OCT site and targeted inactivation of Hoxa1 both impair the response of the b1-ARE to retinoic acid in transgenic mice. Our results show that Hoxa1 is the primary mediator of the response of b1-ARE to retinoic acid in vivo and that this function is dependent on the binding of SOX/OCT heterodimers to the b1-ARE. These results uncover novel functional differences between Hox paralogs and their modulators.

Animals↗

PubMed2001

Synergy between Hoxa1 and Hoxb1: the relationship between arch patterning and the generation of cranial neural crest.

Hoxa1 and Hoxb1 have overlapping synergistic roles in patterning the hindbrain and cranial neural crest cells. The combination of an ectoderm-specific regulatory mutation in the Hoxb1 locus and the Hoxa1 mutant genetic background results in an ectoderm-specific double mutation, leaving the other germ layers impaired only in Hoxa1 function. This has allowed us to examine neural crest and arch patterning defects that originate exclusively from the neuroepithelium as a result of the simultaneous loss of Hoxa1 and Hoxb1 in this tissue. Using molecular and lineage analysis in this double mutant background we demonstrate that presumptive rhombomere 4, the major site of origin of the second pharyngeal arch neural crest, is reduced in size and has lost the ability to generate neural crest cells. Grafting experiments using wild-type cells in cultured normal or double mutant mouse embryos demonstrate that this is a cell-autonomous defect, suggesting that the formation or generation of cranial neural crest has been uncoupled from segmental identity in these mutants. Furthermore, we show that loss of the second arch neural crest population does not have any adverse consequences on early patterning of the second arch. Signalling molecules are expressed correctly and pharyngeal pouch and epibranchial placode formation are unaffected. There are no signs of excessive cell death or loss of proliferation in the epithelium of the second arch, suggesting that the neural crest cells are not the source of any indispensable mitogenic or survival signals. These results illustrate that Hox genes are not only necessary for proper axial specification of the neural crest but that they also play a vital role in the generation of this population itself. Furthermore, they demonstrate that early patterning of the separate components of the pharyngeal arches can proceed independently of neural crest cell migration.

Animals↗

PubMed2000

Retinoid signalling and hindbrain patterning.

Retinoid signalling has been implicated in regulating a wide variety of processes in vertebrate development. Recent advances from analyses on the synthesis, degradation and distribution of retinoids in combination with functional analysis of signalling components have provided important insights into the regulation of patterning the nervous system and the hindbrain in particular.

Animals↗

PubMed1998

Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning.

In the developing vertebrate hindbrain Hoxa1 and Hoxb1 play important roles in patterning segmental units (rhombomeres). In this study, genetic analysis of double mutants demonstrates that both Hoxa1 and Hoxb1 participate in the establishment and maintenance of Hoxb1 expression in rhombomere 4 through auto- and para-regulatory interactions. The generation of a targeted mutation in a Hoxb1 3' retinoic acid response element (RARE) shows that it is required for establishing early high levels of Hoxb1 expression in neural ectoderm. Double mutant analysis with this Hoxb1(3'RARE) allele and other targeted loss-of-function alleles from both Hoxa1 and Hoxb1 reveals synergy between these genes. In the absence of both genes, a territory appears in the region of r4, but the earliest r4 marker, the Eph tyrosine kinase receptor EphA2, fails to be activated. This suggests a failure to initiate rather than maintain the specification of r4 identity and defines new roles for both Hoxb1 and Hoxa1 in early patterning events in r4. Our genetic analysis shows that individual members of the vertebrate labial-related genes have multiple roles in different steps governing segmental processes in the developing hindbrain.

Animals↗

PubMed1998

Hoxa1 and Hoxb1 synergize in patterning the hindbrain, cranial nerves and second pharyngeal arch.

The analysis of Hoxa1 and Hoxb1 null mutants suggested that these genes are involved in distinct aspects of hindbrain segmentation and specification. Here we investigate the possible functional synergy of the two genes. The generation of Hoxa1(3'RARE)/Hoxb1(3'RARE) compound mutants resulted in mild facial motor nerve defects reminiscent of those present in the Hoxb1 null mutants. Strong genetic interactions between Hoxa1 and Hoxb1 were uncovered by introducing the Hoxb1(3'RARE) and Hoxb1 null mutations into the Hoxa1 null genetic background. Hoxa1(null)/Hoxb1(3'RARE) and Hoxa1(null)/Hoxb1(null )double homozygous embryos showed additional patterning defects in the r4-r6 region but maintained a molecularly distinct r4-like territory. Neurofilament staining and retrograde labelling of motor neurons indicated that Hoxa1 and Hoxb1 synergise in patterning the VIIth through XIth cranial nerves. The second arch expression of neural crest cell markers was abolished or dramatically reduced, suggesting a defect in this cell population. Strikingly, the second arch of the double mutant embryos involuted by 10.5 dpc and this resulted in loss of all second arch-derived elements and complete disruption of external and middle ear development. Additional defects, most notably the lack of tympanic ring, were found in first arch-derived elements, suggesting that interactions between first and second arch take place during development. Taken together, our results unveil an extensive functional synergy between Hoxa1 and Hoxb1 that was not anticipated from the phenotypes of the simple null mutants.

Animals↗

PubMed1998

Segmentation and specification in the branchial region of the head: the role of the Hox selector genes.

Hox genes are segmentally expressed in the developing vertebrate hindbrain, neural crest cells and pharyngeal arches suggesting an important role in patterning these structures. Here we discuss the cellular and molecular mechanisms controlling segmentation and specification in the branchial region of the head. In addition, based on the recent phenotypical and molecular analysis of loss-of-function mutants in the mouse, we speculate that Hox genes may act like Drosophila selector genes in this system.

Animals↗

PubMed1997

Role of Hoxa-2 in axon pathfinding and rostral hindbrain patterning.

Segmentation plays an important role in neuronal diversification and organisation in the developing hindbrain. For instance, cranial nerve branchiomotor nuclei are organised segmentally within the basal plates of successive pairs of rhombomeres. To reach their targets, motor axons follow highly stereotyped pathways exiting the hindbrain only via specific exit points in the even-numbered rhombomeres. Hox genes are good candidates for controlling this pathfinding, since they are segmentally expressed and involved in rhombomeric patterning. Here we report that in Hoxa-2(-/-) embryos, the segmental identities of rhombomere (r) 2 and r3 are molecularly as well as anatomically altered. Cellular analysis by retrograde dye labelling reveals that r2 and r3 trigeminal motor axons turn caudally and exit the hindbrain from the r4 facial nerve exit point and not from their normal exit point in r2. Furthermore, dorsal r2-r3 patterning is affected, with loss of cochlear nuclei and enlargement of the lateral part of the cerebellum. These results point to a novel role for Hoxa-2 in the control of r2-r3 motor axon guidance, and also suggest that its absence may lead to homeotic changes in the alar plates of these rhombomeres.

Animals↗

PubMed1995

Analysis of the chicken GPAT/AIRC bidirectional promoter for de novo purine nucleotide synthesis.

GPAT and AIRC encode two enzymes that catalyze steps 1 and 6 plus 7, respectively, of the de novo purine biosynthetic pathway. The chicken genes are closely linked and divergently transcribed from an approximately 230-base pair intergenic region. The promoter was scanned by deletion mutagenesis in a bireporter vector that allowed assay of transcriptional activity in both directions in transfected HepG2 and chicken LMH cells. Three classes of deletions were obtained: those affecting bidirectional transcription, those predominantly affecting GPAT transcription, and those predominantly affecting AIRC transcription. Defects in bidirectional transcription resulted from removal of an initiator-like element overlapping the AIRC transcription start site, as well as deletions removing a series of GC and CCAAT boxes from the AIRC proximal half of the promoter and a CCAAT-containing segment from the GPAT side. Several regions in the GPAT proximal half of the promoter, including an octamer-like motif downstream from the transcription start site, were required predominantly for GPAT expression. Evidence for interaction of HeLa nuclear proteins with some of these sites was obtained by gel retardation, DNase I, and methylation interference assays. Overall, the results showed that the intergenic region is an integrated bidirectional promoter and that a novel initiator-like element plays a central role in coordinating expression of the divergently transcribed AIRC and GPAT genes.

Amidophosphoribosyltransferase↗

PubMed1994

Two genes for de novo purine nucleotide synthesis on human chromosome 4 are closely linked and divergently transcribed.

A cDNA encoding human glutamine phosphoribosylpyrophosphate amidotransferase for step one in de novo purine nucleotide synthesis was cloned, sequenced, and expressed in Chinese hamster ovary cells to yield functional enzyme. Enzyme function was dependent upon removal of an 11-amino-acid propeptide. A mutant enzyme having three propeptide amino acid replacements was not processed and was not active. The human genes GPAT, encoding the amidotransferase, and AIRC, encoding a bifunctional enzyme for steps six and seven in the pathway, were cloned and characterized. GPAT and AIRC are closely linked and divergently transcribed from an intergenic region of approximately 625 base pairs. Expression of a luciferase reporter from the GPAT promoter was approximately 3-4-fold higher than from the AIRC promoter. The GPAT gene was mapped to the q12 region of chromosome 4.

Amidophosphoribosyltransferase↗

PubMed1993

Coexpression of two closely linked avian genes for purine nucleotide synthesis from a bidirectional promoter.

Two avian genes encoding essential steps in the purine nucleotide biosynthetic pathway are transcribed divergently from a bidirectional promoter element. The bidirectional promoter, embedded in a CpG island, directs coexpression of GPAT and AIRC genes from distinct transcriptional start sites 229 bp apart. The bidirectional promoter can be divided in half, with each half retaining partial activity towards the cognate gene. GPAT and AIRC genes encode the enzymes that catalyze step 1 and steps 6 plus 7, respectively, in the de novo purine biosynthetic pathway. This is the first report of genes coding for structurally unrelated enzymes of the same pathway that are tightly linked and transcribed divergently from a bidirectional promoter. This arrangement has the potential to provide for regulated coexpression comparable to that in a prokaryotic operon.

Amidophosphoribosyltransferase↗

PubMed1991

Anti-inflammatory and immunomodulating effects of the novel agent gamma-(2-aminoethylamino)-2-butyrothienone. 1st communication: inhibitory effects on mouse paw edema.

A series of 4-hydroxy-(or-amino)-ethyl-amino butyrophenones or butyrothienones were synthesized. For detail studies on antiinflammatory effects, gamma-(2-aminoethylamino)-2-butyrothienone (gamma-ABT) was chosen as representative of these new non-steroidal anti-inflammatories (NSAID). The effect on mouse paw edema induced by various phlogistic agents was first investigated. The inhibitory effect of gamma-ABT on carrageenin-induced edema was remarkable and nearly equal to that of indometacin. Similarly to indometacin, gamma-ABT inhibited the early and late stage of yeast-induced edema in contrast to concanavalin A (Con A) induced edema which was only inhibited by gamma-ABT. Both the above induced edema are supposed to be unrelated to prostaglandins in the rat system. gamma-ABT displayed an inhibitory effect on nystatin-induced edema similar to indometacin suggesting that gamma-ABT has significant membrane stabilizing action and a strong blocking action on synthesis of prostaglandins. gamma-ABT inhibited as well the sustained edema induced by mustard. In conclusion gamma-ABT is an effective agent not only on acute but also on subacute and chronic inflammation and its mode of action appears similar to other NSAID. gamma-ABT posses in addition the advantage of antioxidant activity which is not shared by selective cyclooxygenase inhibitors and thus should be potentially effective in autoimmune diseases.

Animals↗

PubMed1991

Anti-inflammatory and immunomodulating effects of the novel agent gamma-(2-aminoethylamino)-2-butyrothienone. 2nd communication: inhibitory effect on rat adjuvant induced disease.

The effect of the novel agent gamma-(2-aminoethylamino)-2-butyrothienone (gamma-ABT) on local and systemic changes of rats with adjuvant induced disease (AID) was investigated, gamma-ABT showed potent inhibitory effect on adjuvant primary inflammation and almost totally inhibited the secondary lesions. gamma-ABT improved the changes in lymphoid organ weight except of the thymus. gamma-ABT improved the change in albumin/globulin ratio, which is a parameter of systemic inflammatory reaction but did not improve the body weight gain in AID and normal rats. In addition gamma-ABT did not affect directly T or B lymphocytes as shown by the lymphocyte responses to mitogen (Con A) and a T cell dependent antigen (SRBC) but rather inhibited the suppressor cells found in AID. Although structurally gamma-ABT is completely different from known non-steroidal anti-inflammatories, immunosuppressive drugs behave to a great extent like them.

Adjuvants, Immunologic↗

PubMedDate not supplied

The anti-inflammatory effect of opioids.

The anti-inflammatory activity of two novel opioids PM and PO as well as of pethidine was studied. The mouse paw edema, induced by various phlogistic agents, was significantly inhibited after the administration of opioids, fact that was independent of their antioxidant properties. The anti-inflammatory action of the above opioids was not reversed by naloxone. These results suggest that a variety of complex regulatory activities may be performed by opioid agonists via naloxone-sensitive or naloxone insensitive receptors on inflammatory cells, directly or indirectly by the inhibition of cytokines and mediators involved in inflammation.

Animals↗