Biomedical subjects

J M Wilson

Publications and source records attributed to J M Wilson.

At least 253 records · Page 14Linked to original sources

PubMed1995

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.

Adenoviruses, Human↗

PubMed1995

Design and evaluation of a sterile compounding facility.

The design and evaluation of a sterile compounding center for a large community teaching hospital are described. The new sterile products area was redesigned to improve efficiency by minimizing staff travel and to incorporate recommendations of the ASHP Technical Assistance Bulletin on Quality Assurance for Pharmacy-prepared Sterile Products. The design approach combined strategic planning with master facilities planning. The process began with a systems analysis, followed by the development of a functional program (a comprehensive list of design specifications). Travel studies were performed before and after renovation to determine gains in efficiency; technician travel decreased 29% and pharmacist travel, 42%. The new facility design met the specifications in the functional program and appeared to comply with all recommendations in the ASHP document for all risk levels of preparations.

Drug Compounding↗

PubMed1995

Molecular characterization of an apical early endosomal glycoprotein from developing rat intestinal epithelial cells.

The apical endosomal compartment is thought to be involved in the sorting and selective transport of receptors and ligands across polarized epithelia. To learn about the protein components of this compartment, we have isolated and sequenced a cDNA that encodes a glycoprotein that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. The deduced amino acid sequence predicts a protein of 1216 amino acids with a molecular mass of 133,769 Da. The deduced amino acid sequence together with amino-terminal amino acid sequencing indicate that there is a cleaved 21-amino acid signal sequence at the NH2-terminal portion of the molecule. There is a single hydrophobic region near the carboxyl terminus that has the characteristics of a membrane-spanning domain and a 36-amino acid cytoplasmic tail. We have found that the major form of this protein in intestinal epithelial cells has a molecular mass of 55-60 kDa, which is significantly smaller than the size predicted from the cDNA sequence, suggesting that the protein is synthesized as a large precursor and processed to the smaller form. The smaller form remains associated with the membrane, however, possibly through noncovalent association with the transmembrane portion of the molecule or with another membrane protein. The extracytoplasmic domain is cysteine-rich, with three cysteine-rich repeats that are similar to cysteine repeats present in several receptor proteins. However, there is no other significant similarity to other proteins in the GenBank. The cytoplasmic tail contains a possible internalization motif and several consensus motifs for serine/threonine kinases. Northern blot analysis suggests a single abundant message, and Southern blot analysis is consistent with a single gene and the absence of pseudogenes for this unique endosomal protein.

Amino Acid Sequence↗

PubMed1995

Activated clotting times in acute coronary syndromes and percutaneous transluminal coronary angioplasty.

A discrete fall in the ACT (activated coagulation time) has been observed in patients with known activation of the coagulation cascade. Injury to the coronary artery resulting in thrombin activation, whether spontaneous as in the case of acute myocardial infarction or planned as with percutaneous transluminal coronary angioplasty (PTCA), may therefore be reflected in a change in ACT values. We reviewed the records of patients undergoing PTCA at St. Luke's Episcopal Hospital/Texas Heart Institute from January 1990 through December 1992 for information regarding ACT values and clinical events. A total of 469 patients, whose record contained adequate information for study inclusion, were divided into four separate groups: acute myocardial infarction (group I, n = 62), unstable angina with heparin therapy that was withdrawn at least 4 hr prior to PTCA (group II, n = 102), unstable angina with heparin therapy continued until the time of PTCA (group III, n = 154), and stable angina undergoing elective PTCA (group IV, n = 151). Heparin was discontinued 12-15 hr after the procedure in all but group I where anticoagulation was often maintained up to 72 hr. ACT values were measured prior to the PTCA procedure (baseline), after the initial heparin bolus of 10,000 U (postheparin) and approximately 12-18 hr after the procedure (heparin withdrawal).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1995

Single- and multiple-dose pharmacokinetics of dezocine in patients with acute or chronic pain.

The pharmacokinetic properties of dezocine were examined in 15 patients with acute or chronic pain. In 3 groups of 5 patients each, serum levels were determined at various intervals after single intravenous doses of 5, 10, and 20 mg. After these single doses, dezocine was very rapidly distributed (mean t1/2 alpha less than 2 minutes), and then rather rapidly eliminated (mean t1/2 beta about 4 hours); the apparent volume of distribution was large (mean Vz beta about 6 L/kg) as was the total clearance (mean CL about 1.5 L/h/kg). In 2 groups of 5 patients each, serum levels were determined after the first and third of 3 intravenous doses of 5 or 20 mg given at 3-hour intervals. The pharmacokinetic parameters after these multiple doses were consistent with those after the single doses. Although some observations were suggestive, there was no unequivocal evidence that the pharmacokinetics were dose-related. In 7 serum samples containing dezocine at concentrations ranging from 12.8 to 522 ng/mL, the mean (+/- SE) proportion of dezocine bound to protein was 91.6 +/- 0.8%.

Acute Disease↗

PubMed1995

Characterization of stem cells in human airway capable of reconstituting a fully differentiated bronchial epithelium.

The epithelia of the lung are complex structures that play an important role in normal lung physiology and are often involved in the pathophysiology of pulmonary diseases. The dynamics of cell turnover, lineage, and differentiation within these epithelia are complex and poorly understood. We have coupled the technique of retrovirus-mediated gene transfer with a xenograft model of proximal human airway to evaluate pathways of cellular proliferation and differentiation in human bronchial epithelia. Primary isolates of human bronchial epithelial cells (HBECs) were infected with mixtures of recombinant retroviruses expressing different reporter genes and seeded into denuded rat trachea, which were implanted subcutaneously into athymic mice. The HBECs were allowed to regenerate for four weeks in xenografts, which were then explanted. Clonal expansion of individual retrovirus-marked cells in the regenerated human bronchial epithelium was detected as clusters of transgene-expressing cells. Clone size varied with seeding density, resulting in the largest clones comprising 10(3)-10(4) cells. A substantial number of clones showed transgene expression in basal as well as differentiated columnar cells, a finding that appeared independent of clone size. These studies demonstrate the existence of a cell type within the human bronchial epithelium that is capable of extensive self-renewal and pluripotent development. Further characterization of these potential stem cells will be important in defining pathogenesis of pulmonary diseases and in developing novel approaches to treatment such as gene therapy.

Animals↗

PubMed1995

Experimental fetal tracheal ligation and congenital diaphragmatic hernia: a pulmonary vascular morphometric analysis.

The authors have previously shown that fetal tracheal ligation (TL) reverses the pulmonary hypoplasia in experimental diaphragmatic hernia (DH) by accelerating fetal alveolar growth. The purpose of this study was to determine if growth of the accompanying macroscopic and microscopic pulmonary vasculature is also accelerated. Eighteen fetal lambs were divided into three experimental groups: diaphragmatic hernia (DH), DH and simultaneous tracheal ligation (DH/TL), and sham-operated controls (C). Animals were delivered near term, the lungs retrieved, and pulmonary capillary growth (5 to 50 microns in diameter) evaluated by standard morphometric techniques. Capillary ultrastructure was evaluated by electron microscopy. Nine additional fetal lambs of the same gestational age were equally divided into the same three groups and their lungs analyzed by pulmonary arteriography for evaluation of large vessel growth (< 100-microns diameter). Computer digital analysis of angiogram lung slices showed that the total area of large vessels was increased in DH/TL lungs when compared with DH lungs and decreased in DH lungs when compared with C lungs (P = .003); however, the ratio of large vessel area per unit of lung area was similar in all groups. Microscopic morphometry of the capillary bed showed that the total number of capillaries was increased in DH/TL lungs over both DH and C lungs (P = .0001); however, the number of capillaries per alveolus (cap/alv) was similar in all groups. In DH/TL lungs, electron microscopy showed normal capillary wall structure and normal thickness of the capillary-alveolar interface, whereas in DH lungs, capillary structure was abnormal and the capillary-alveolar interface was thickened.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiography↗

PubMed1995

Fate of the reconstructed carotid artery after extracorporeal membrane oxygenation.

Reconstruction of the right common carotid artery has been shown to be feasible in neonates after extracorporeal membrane oxygenation (ECMO). However, the long-term outcome after carotid artery reconstruction (CAR) remains unknown. The purpose of this study was to evaluate the natural progression of the anastomotic site after CAR. Between February 1990 and June 1993, 201 patients received ECMO. All veno-arterial (VA) ECMO patients (n = 172) were considered candidates for reconstruction unless a significant neurological event (ie, intracranial hemorrhage, stroke) had occurred; the duration of ECMO exceeded 10 days, making carotid mobilization difficult; or the patient's prognosis was deemed poor. Reconstruction was performed by excising the arteriotomy site, followed by primary end-to-end anastomosis. Reconstruction was abandoned and the artery ligated if an intimal flap, arterial thrombosis, or excessive tension was encountered. After reconstruction all patients had early carotid ultrasonography and either head computed tomography (CT) or magnetic resonance imaging (MRI). Subsequent ultrasound examinations were performed at approximately 6-month intervals. Diameter index (DI) (a measure of anastomotic narrowing) was calculated using ultrasound by dividing the anastomotic diameter by the diameter of the carotid artery 5 mm proximal to the anastomosis. Forty-three of 172 VA ECMO patients (25%) had successful reconstruction. Long-term follow-up data were available on 27 patients. These 27 patients had 39 ultrasound examinations, with an average follow-up time of 7.3 months (range, 4 days to 29 months). All carotid arteries were patent. Linear regression analysis showed significant improvement in the DI with time (P = .0001, r2 = .382).(ABSTRACT TRUNCATED AT 250 WORDS)

Anastomosis, Surgical↗

PubMed1995

Triple therapy in cadaveric renal transplantation: role of induction cyclosporine and targeted levels to avoid rejection.

The updated data on 61 consecutive cadaveric transplants performed at our institution from 1987 to 1990 (followup 31 to 82 months, median 54 months) were analyzed with emphasis on cyclosporine monitoring and long-term results. All patients received triple therapy with cyclosporine induction, azathioprine and prednisone regardless of graft function, and they were preferentially placed on the calcium blocker nifedipine. We monitored 12-hour cyclosporine trough levels in whole blood using high performance liquid chromatography and the dose was adjusted to maintain levels at 150 ng./ml. or greater for the first 3 months. In 17 of 61 patients (28%) 22 rejection episodes occurred and 20 nephrotoxicity episodes occurred in 17 of 61 patients (28%). There was no significant difference in the mean cyclosporine levels among 32 rejection, nonrejection, nephrotoxic and nonnephrotoxic cases at any interval. Rejection occurred by 1 month in 13 (76%) and by 3 months in 15 (88%) of 17 patients. Comparisons were made in the first month to define the desirable cyclosporine levels by calculating the mean cyclosporine only within 10 to 14 days of rejection or nephrotoxicity events. The mean cyclosporine level before rejection was significantly lower than that for nephrotoxicity (188 +/- 113 versus 304 +/- 62 ng./ml., p < 0.01). The median cyclosporine level for first month rejection was also significantly lower than that for nonrejection (156 versus 218 ng./ml., p < 0.05) and it was significantly greater for nephrotoxicity versus nonnephrotoxicity (272 versus 218 ng./ml., p < 0.05). Of 13 rejections in the first month 10 (77%) were associated with mean levels of less than 210 ng./ml. Actuarial graft survival at 1, 3 and 5 years was 93.4%, 87.8% and 78.5%, respectively. The 3-year graft survival was significantly worse for patients who experienced acute rejection episodes versus those who did not (68.8% versus 96.7%, p < 0.05) but it was not different for nephrotoxic versus nonnephrotoxic groups (85.6% versus 79.6%). Long-term function was not influenced by the occurrence of acute nephrotoxicity events. These findings confirm the efficacy of triple therapy with induction cyclosporine in cadaveric transplantation, yielding improved short-term and intermediate graft survival without any adverse effects on long-term graft function. Specific cyclosporine level monitoring is invaluable, particularly initially, with high target levels of 200 ng./ml. or greater. The use of calcium blockers may have allowed higher cyclosporine dosing in the first 3 months, mitigating against cyclosporine associated chronic nephrotoxicity.

Actuarial Analysis↗

PubMed1995

Gene therapy in a xenograft model of cystic fibrosis lung corrects chloride transport more effectively than the sodium defect.

We have developed a model of gene therapy for cystic fibrosis (CF) lung disease, based on growth of human CF bronchial xenografts in nu/nu mice. We now report an evaluation of the primary abnormalities in CF lung epithelia--defective Cl secretion and Na hyperabsorption--in xenografts following adenovirus-mediated gene transfer. In vivo infection of CF xenografts with a cystic fibrosis transmembrane regulator (CFTR) recombinant adenovirus, at a multiplicity of infection equal to 100, was sufficient to reconstitute near normal levels of cAMP-stimulated Cl transport, despite transducing only 5% of cells in the pseudostratified epithelium. Correction in sodium hyperabsorption was partial and variable. These experiments define aspects of adenovirus-mediated gene therapy relevant to CF protocols based on intrapulmonary genetic reconstitution.

Absorption↗

PubMed1995

Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung.

Enthusiasm for the use of recombinant adenoviruses in gene therapy has been tempered by the problematic immune responses that develop to the virus and virus-infected cells. Humoral immune responses to the input viral proteins generate neutralizing antibodies that thwart attempts to effectively administer the therapy more than once. Previous studies in murine models of gene therapy for cystic fibrosis (CF) have shown that the formation of adenoviral antibodies of the IgA subtype, a process that is dependent on T helper cells of the TH2 subset, contributes to a block in gene transfer that occurs following a second administration of virus. We show in this report that coadministration of interferon-gamma (IFN-gamma) (or interleukin-12, which activates TH1 cells to secrete IFN-gamma) with the recombinant adenovirus into the airway of C57BL/6 mice diminishes the activation of TH2 cells and formation of neutralizing antibody, allowing for efficient readministration of recombinant virus. This suggests a strategy for gene therapy of CF in which administration of a short-acting immune modulator at the time of gene therapy may be sufficient to overcome the problems of humoral immunity.

Adenoviridae↗

PubMed1995

Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study.

This paper describes a preclinical toxicology study designed to investigate the biological efficacy and safety profile of second-generation adenovirus for CFTR gene transfer into the baboon lung. This second-generation virus is deleted of E1 and contains a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein. Two distinct projects were undertaken. Group A animals received a first-generation adenovirus (i.e., deleted of E1) in an upper lobe at the time a second-generation virus was instilled into the contralateral upper lobe. The goal of study A was to compare the biology of each construct directly and to determine if an immune response to the first-generation virus affected the performance of the second-generation virus. Group B animals received a lacZ second-generation virus in an upper lobe at the same time the CFTR second-generation virus was instilled in the other upper lobe. Necropsies were performed 4 or 21 days after gene transfer and tissues were evaluated for recombinant gene expression and histopathology. Using a second-generation adenovirus, recombinant gene stability was prolonged and associated with a diminished level of perivascular inflammation as compared to first-generation vectors. Markedly diminished levels of hexon protein were present in tissues infected with second-generation as compared to first-generation virus. No evidence of viral shedding was evident. Furthermore, coadministration of first- and second-generation adenovirus did not affect the stability of transgene expression from the second-generation virus. These data suggest that second-generation adenoviral vectors provide an improved gene delivery vehicle, and thus may be useful in gene therapy for cystic fibrosis.

Adenoviridae↗

PubMed1995

Treatment of experimental human mesothelioma using adenovirus transfer of the herpes simplex thymidine kinase gene.

OBJECTIVE: The authors demonstrate the ability of an adenovirus vector expressing the herpes simplex thymidine kinase (HSVtk) gene to treat human malignant mesothelioma growing within the peritoneal cavity of severe combined immunodeficient (SCID) mice. BACKGROUND DATA: Introduction of the HSVtk gene into tumor cells renders them sensitive to the antiviral drug ganciclovir (GCV). This approach has been used previously to treat experimental brain tumors. Although malignant mesothelioma is refractory to current therapies, its localized nature and the accessibility of the pleural space make it a potential target for a similar type of in vivo gene therapy using adenovirus. METHODS: An adenovirus containing the HSVtk gene (Ad.RSVtk) was used to transduce mesothelioma cells in vitro. These cells were then injected into the flanks of SCID mice. Ad.RSVtk was also injected directly into the peritoneal cavity of SCID mice with established human mesothelioma tumors. Mice were subsequently treated for 7 days with GCV at a dose of 5 mg/kg. RESULTS: Mesothelioma cells transduced in vitro with Ad.RSVtk formed nodules when injected in the subcutaneous tissue. These tumors could be eliminated by the administration of GCV, even when as few as 10% of cells were transduced to express HSVtk (bystander effect). Administration of Ad.RSVtk into the peritoneal space of animals with established multifocal human mesothelioma followed by GCV therapy resulted in the eradication of macroscopic tumor in 90% of animals and microscopic tumor in 80% of animals when evaluated after 30 days. The median survival of animals treated with Ad.RSVtk/GCV was significantly longer than that of control animals treated with similar protocols. CONCLUSION: These results indicate that an adenoviral vector containing the HSVtk gene is effective in treating established malignant mesothelioma in an in vivo setting and raise the possibility of using adenovirus transfer of HSVtk for clinical trials in mesothelioma and other localized tumors.

Adenoviruses, Human↗

PubMed1995

Identification of a marker antigen for the endocytic stage of intestinal development in rat, sheep, and human.

Vacuolated enterocytes are highly endocytic epithelial cells present in intestines of diverse mammalian species during neonatal and/or fetal development. Using monoclonal antibodies raised against membrane fractions, we previously identified a 55-61 kd membrane glycoprotein that is restricted to apical endosomal tubules of vacuolated enterocytes in fetal and suckling rats. To determine whether this cell-specific antigen is present in vacuolated enterocytes of fetal sheep or humans, the endosomal antigen was immuno-affinity purifed from rats and used to generate and purify specific rabbit polyclonal antibodies. Light microcopic and electron microscopic immunocytochemistry showed that antigens cross-reactive with the rat endosomal antigen are present in vacuolated enterocytes of fetal sheep and fetal human small intestine and are restricted to apical endosomal tubules in these cells. Immunoblot analysis of tissue extracts from fetal human intestines showed antigen(s) at 55 and 60 kd, as well as a major form at 130 kd. Cross-reactive antigen(s) from fetal sheep intestines appeared as 42- and 50-kd bands. Although the molecular identities of the sheep and human antigens are not yet established, these results show that these antigens can serve as markers for the endocytic state of intestinal developmemt in humans as well as other mammals.

Animals↗

PubMed1995

SB 205952, a novel semisynthetic monic acid analog with at least two modes of action.

The biological properties of SB 205952, a nitrofuryl oxazole derivative of monic acid, differ from those of the closely related antibacterial agent mupirocin. Compared with mupirocin, SB 205952 has increased antimicrobial potency, an extended spectrum including mupirocin-resistant staphylococci, and rapid bactericidal activity. SB 205952, like mupirocin, is a potent inhibitor of bacterial isoleucyl-tRNA synthetase (IRS) in mupirocin-susceptible organisms but does not inhibit IRS from mupirocin-resistant staphylococci, indicating that SB 205952 has more than one mechanism of action. SB 205952 rapidly inhibits protein, RNA, and DNA syntheses in mupirocin-susceptible and mupirocin-resistant staphylococci. In each case, the effect on RNA synthesis is relaxed by treatment with chloramphenicol, indicating that inhibition of RNA synthesis is probably a secondary consequence of stringent control. It is proposed that SB 205952 possesses one or more mechanisms of action in addition to IRS inhibition, probably mediated by its nitrofuryl component.

Anti-Bacterial Agents↗

PubMed1995

Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway.

Recombinant adenoviruses are being evaluated for gene therapy of cystic fibrosis lung disease with the goal of reconstituting the expression of the cystic fibrosis transmembrane conductance regulator in pulmonary epithelia by direct administration of the virus into the airway. The therapeutic potential of recombinant adenoviruses is limited in part by the relative inefficiency by which gene transfer occurs. This study uses a human bronchial xenograft model to study adenovirus infection in the human airway in an attempt to define the molecular events that limit gene transfer. Our studies of the human airway confirm previous observations of cell lines that have indicated a two-step process for adenovirus entry, which begins with the binding of the virus to the cell through the fiber protein and continues with internalization via interactions among cellular integrins and an RGD motif (Arg-Gly-Asp) in the penton base. Furthermore, the level of maturity of the epithelia in xenografts has a major impact on gene transfer. Undifferentiated epithelia express high levels of alpha v beta 5 integrins and are easily infected with recombinant adenoviruses; gene transfer is completely inhibited with excess fiber and partially inhibited with RGD peptide and alpha v beta 5 integrin antibody. Pseudostratified epithelia do not express alpha v beta 5 integrin in differentiated columnar cells and are relatively resistant to adenovirus-mediated gene transfer; what little gene transfer occurs is inhibited by fiber but not by RGD peptide or alpha v beta 5 integrin antibody. These studies suggest that the expression of integrins in human airway epithelia limits the efficiency of gene transfer with recombinant adenoviruses. However, low-level gene transfer can occur in fully mature epithelia through alpha v beta 5 integrin-independent pathways.

Adenoviridae↗

PubMed1995

Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.

Recombinant adenoviruses are an attractive vehicle for gene therapy to the lung in the treatment of cystic fibrosis (CF). First-generation viruses deleted of E1a and E1b transduce genes into airway epithelial cells in vivo; however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. In this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive transfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. Our studies indicate that major histocompatibility complex class I-restricted CD8+ cytotoxic T lymphocytes are activated in response to newly synthesized antigens, leading to destruction of virus infected cells and loss of transgene expression. Major histocompatibility complex class II-associated presentation of exogenous viral antigens activates CD4+ T-helper (TH) cells of the TH1 subset and, to a lesser extent, of the TH2 subset. CD4+ cell-mediated responses are insufficient in the absence of cytotoxic T cells to completely eliminate transgene containing cells; however, they contribute to the formation of neutralizing antibodies in the airway which block subsequent adenovirus-mediated gene transfer. Definition of immunological barriers to gene therapy of cystic fibrosis should facilitate the design of rational strategies to overcome them.

Adenoviridae↗

PubMed1995

Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

3T3 Cells↗