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A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to conventional SPPS reaction vessels, which drain solvent and byproducts via a frit located at the bottom of the vessel, the presented apparatus employs a gas dispersion tube under vacuum to remove solvent, byproducts, and excess reagents. The same gas dispersion tube supplies nitrogen gas agitation of the SPPS beads during the reaction steps of coupling and deprotection. Microwave heating is beneficial for SPPS couplings of sterically hindered residues, such as alpha-aminoisobutyric acid (Aib), an alpha,alpha-dialkylated amino acid residue. This home-built apparatus has been used to prepare, via manual Fmoc SPPS methods, heptameric and octameric peptides dominated by the Aib residue, which is notoriously difficult to couple under standard room temperature conditions and reagents. Further, typical commercial microwave SPPS reactors are dedicated exclusively to SPPS synthesis rendering them inaccessible to non-SPPS users. In contrast, the presented apparatus preserves the versatility of the microwave reactor for conventional microwave acceleration of chemical reactions, as the apparatus is trivially removed from the commercial microwave reactor.

Statement of Problem: Clinical adjustment of a lithium disilicate glass-ceramic (LDGC) restoration may necessitate its return to the laboratory for additional firing. Evidence of how the intaglio surface should be re-etched after internal adjustment, or after refiring, is lacking., Purpose: The purpose of this in vitro study was to investigate the effects of different sequences of etching, refiring, diamond rotary instrument adjustment, airborne-particle abrasion, and re-etching on the microstructure and surface roughness of the intaglio surface of heat-pressed LDGCs., Material and Methods: Heat-pressed LDGC specimens were ground with abrasive paper to produce a uniformly flat surface. The groups (n=3) were subjected to different combinations of etching, refiring, diamond rotary instrument adjustment, airborne-particle abrasion, and re-etching. X-ray diffraction was used to characterize the crystalline phases. Scanning electron microscopy and surface profilometry were used to characterize the microstructure and surface roughness., Results: Qualitative differences were observed in the surface texture of specimens etched for different periods. Excessive etching revealed more of the underlying lithium disilicate crystallites and caused surface pitting for the longest etching period studied. Refiring altered the surface condition but did not completely remove the texture created by the original etching. Diamond rotary instrument adjustment resulted in appreciable surface damage and a higher mean value of measured surface roughness (with or without re-etching) than the other groups. Airborne-particle abrasion caused embedding of particles in the specimen surface, likely corresponding to the abrasion media, although this process resulted in qualitatively less surface damage than diamond rotary instrument adjustment., Conclusions: Excessive etching, refiring, and adjustment by airborne-particle abrasion or diamond rotary instrument result in qualitative changes in surface condition. Adjustment by diamond rotary instrument results in appreciable surface damage., (Copyright © 2019 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

The literature is sparse regarding treatment of burn scar equinus contracture, with focus primarily on staged procedures, serial casting, and gradual correction using external fixation in combination with soft-tissue procedures. This case study describes a single-stage ambulatory approach for late-stage correction of burn scar equinus contracture associated with toe walking. A case report is presented of an 11-year-old male with focus on procedure selection, surgical technique, and 12-month follow-up results. Surgery involved a single-stage approach with open Achilles lengthening, in addition to multiple skin Z-plasty in parallel with immediate protected weightbearing to correct toe walking. Inadequate release of contracture was noted intraoperatively after Achilles lengthening. Full correction was achieved after converting the longitudinal incision into multiple Z-plasty in parallel, with full heel purchase at 2 weeks postoperatively. The patient was completely healed with pain-free range of motion at 6 weeks postoperatively. At 12 months postoperatively, he continued to ambulate normally without overcorrection or recurrence of deformity. This case study describes a late-stage, minimally invasive, single-stage approach to correction of burn scar equinus contracture. The surgical principles and technique are described. Allowance of immediate weightbearing was possible because all other burn wounds were healed at late-stage presentation that avoided the need for gradual correction with external fixation or serial procedures., (Copyright © 2019 the American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.)

Objectives: The aim of this systematic review was to identify from in vitro studies the effect of endodontic access on the fracture resistance and damage around the access cavity of all-ceramic crowns., Data: The articles identified were screened by two reviewers according to inclusion and exclusion criteria. The reference lists of articles advanced to second round screening were hand searched to identify additional potential articles. The risk of bias for the articles was independently performed by two reviewers., Sources: An electronic search was conducted on PubMed/Medline, Web of Science, Scopus and Embase databases with no limitations., Study Selection: 383 articles were identified, of which, eight met the inclusion criteria and formed the basis of this systematic review. Factors investigated in the selected articles included the, presence of microcracks at the access cavity, repair protocol, ceramic type, crown fabrication method, luting agent and grit size of the diamond bur. The risk of bias was deemed to be high for three, medium for two and low for three of the reviewed studies. The high level of heterogeneity across the studies precluded meta-analyses., Conclusion: Based on the currently available scientific evidence, a 'best practice' protocol with regard to improving the fracture resistance of endodontically accessed and repaired all-ceramic crowns cannot be conclusively identified. However, some key factors which potentially impact on the fracture resistance of endodontically accessed and repaired all-ceramic crowns have been isolated. Cautious clinical interpretation of these factors is concluded for the maintenance of the crown as a permanent restoration., Clinical Significance: Key factors which impact on the fracture resistance of endodontically accessed and repaired all-ceramic crowns have been isolated from in vitro studies. Cautious clinical interpretation of these factors is advised for the maintenance of the crown as a permanent restoration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Statement of Problem: The excess material produced after heat pressing a lithium disilicate glass ceramic restoration can be either discarded or reused. The reuse of this material requires that any degradation of the material quality be investigated., Purpose: The purpose of this study was to investigate the number of times that leftover lithium disilicate material can be re-pressed and to determine the effect that repeated use has on material properties., Material and Methods: A large (6.1 g) lithium disilicate ingot (A3.5) was heat pressed to yield a ceramic disk (15 × 1.5 mm) for testing. The leftover material was reused to produce a further 3 disks, with the number of pressings increasing for each specimen. An additional unpressed group was included to investigate the properties before pressing so that, in total, 5 groups were established. Specimens were tested for biaxial flexural strength, Vickers hardness, and fracture toughness. X-ray diffraction was used to characterize the crystalline phase, scanning electron microscopy for the microstructure, and differential scanning calorimetry for the thermal properties., Results: No significant difference was found in the biaxial flexural strength of the groups. The hardness of the material decreased, and no significant difference was seen in fracture toughness with repeated pressings. An increase in grain size was observed with increased pressings. By using x-ray diffraction analysis, lithium disilicate was identified as the main crystal phase, and no difference in crystalline composition was found with repeated processing., Conclusion: This material can be reused while maintaining good mechanical properties and without significantly altering the chemical or crystalline composition in an adverse manner., (Copyright © 2014 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

Objectives: It is proposed that a non-uniform rational B-spline (NURBS) based solid geometric model of a ceramic crown would be a flexible and quick approach to virtually simulate root canal access cavities. The computation of strain components orthogonal to surface flaws generated during the drilling would be an appropriate way of comparing different access cavity configurations., Methods: A μCT scan is used to develop a full 3D NURBS geometric solid model of a ceramic crown. Three different access cavity configurations are created virtually in the geometric model and there are then imported into proprietary finite element software. A linear analysis of the each crown is carried out under appropriate in vivo loading and the results are post-processed to carry out a quantitative comparison of the three configurations, Results: The geometric model is shown to be a flexible and quick way of simulation access cavities. Preliminary indications are that post processed strain results from the finite element analysis are good comparators of competing access cavity configurations., Significance: The generation of geometric solid models of dental crowns from μCT scans is a flexible and efficient methodology to simulate a number of access cavity configurations. Furthermore, advanced post-processing of the primary finite element analysis results is worthwhile as preliminary results indicate that improved quantitative comparisons between different access cavity configurations are possible., (Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Background: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine, belonging to the interleukin-6 family of cytokines, that has been suggested to have positive effects on myogenesis following injury and to minimise dystrophic pathology in mdx mice. Previous reports have suggested that Lif mRNA is up-regulated in the limb and diaphragm muscles of mdx mice, in human cases of dystrophy and acutely following exercise. This study examined expression of Lif mRNA in the quadriceps muscles of mdx and wild-type mice that were either sedentary or allowed to exercise voluntarily for two weeks., Results: Exercise caused a decrease in Lif mRNA expression in wild-type muscle, but this was not the case in mdx muscle. Lif mRNA levels in sedentary mdx mice were similar to those in exercised wild type muscles, and in mdx mice there was no further decrease in levels following exercise. Similar down-regulation of Lif mRNA was observed in the tibialis anterior and diaphragm muscles of mdx mice at three and six weeks of age respectively, compared with wild-type controls. Transcripts for the LIF receptor (Lifr) were also down-regulated in these mdx muscles, suggesting LIF activity may be minimised in dystrophic muscle. However fluorescent immunohistochemical labeling of LIF did not correlate with transcript expression data, as LIF immunoreactivity could not be detected in wild-type muscle, where mRNA expression was high, but was present in dystrophic muscle where mRNA expression was low. This study also described the translocation of membrane proteins, including LIFR, to the nuclei of syncytial muscle cells during differentiation and fusion. In addition this study demonstrates that survival of donor myoblasts injected into dystrophic muscle was enhanced by co-administration of recombinant LIF., Conclusions: This study provides new evidence to support a role for LIF in normal muscle biology in response to exercise. Although expression levels of Lif transcript in mdx muscles were not consistent with previous studies, the detection of LIF protein in mdx muscle but not wild-type muscle supports a role for LIF in dystrophy. This study also provides evidence of the differential localisation of the LIFR, and the potential for anti-inflammatory actions of LIF that promote survival of transplanted myoblasts in dystrophic muscle.*corresponding author: Jason White, Muscular Dystrophy Research Group, Murdoch Childrens Research Institute; email: jasondw@unimelb.edu.au.

This study investigated the effect of surface treatments on the shear bond strength of an auto-polymerising acrylic resin cured to acrylic denture teeth. The surface treatments included a combination of grit-blasting and/or wetting the surface with monomer. Samples were prepared and then stored in water prior to shear testing. The results indicated that the application of monomer to the surface prior to bonding did not influence the bond strength. Grit blasting was found to significantly increase the bond strength.

This article describes a technique for producing a duplicate denture using a visible light-polymerized (VLP) denture base for support prior to processing. A 2-part mold of the original denture was made, and then a sheet of VLP resin was reduced to a thickness of 0.5 mm and adapted to the fitting surface of the mold to create a denture base. The base was polymerized and the remaining features, such as the teeth and polished surfaces of the denture, were reproduced in wax. This technique may be helpful when performing subsequent clinical and laboratory procedures.

Objectives: A series of ionomer glasses based on the formula: 4.5SiO2-1.5P2O5-(X)Al2O3-4.5CaO-0.5CaF2, were investigated where X was varied from 3.0 to 1.5 in order to develop heat pressable dental ceramics., Methods: The glasses were heat-pressed and then subjected to different heat-treatment cycles. The mechanical properties of the glass-ceramics were investigated, specimens were tested for hardness, fracture toughness (indentation method) and flexural strength (biaxial method)., Results: Good mechanical properties were obtained for heat-treatments at lower temperatures (i.e. 1150 degrees C). At intermediate heat-treatment temperatures the glass-ceramics were highly crystalline which did not favor the mechanical properties. There appears to be an inverse relationship between fracture toughness and flexural strength. High fracture toughness values of 2.7 (0.4) MPam0.5 were produced for the X = 2.8 glass heat-treated for 8 h at 1150 degrees C, the flexural strength was lowest for this heat-treatment. High flexural strengths of 194.4 (75.0) MPa were obtained by heat-treating the same glass for 1 h at 1150 degrees C. Increasing the hold time increases crystal size thereby increasing the extent of microcracking in the glass-ceramic thus lowering the flexural strength. Microcracks appear to increase the fracture toughness of the glass-ceramics probably by a crack termination mechanism., Significance: Good flexural strength and high fracture toughness are attainable in this system, but appear to be mutually exclusive in the materials studied. With further investigation this system could provide clinically useful materials.

Objectives: This study investigated a series of ionomer glasses based on the formula: 4.5SiO(2)-1.5P(2)O(5-)(X)Al(2)O(3)-4.5CaO-0.5CaF(2), where X was varied from 3.0 to 1.5. The possibility of processing ionomer glasses using a heat-pressing method for dental restorations was investigated., Methods: A simple flow test was designed to measure the amount of flow the glasses underwent as a result of heat-pressing at 1150 degrees C for different times. Heat-pressed samples of the X=3.0, 2.8, 2.4 and 2.0 glass were further heat-treated for 1 and 4 h at 1150, 1200 and 1250 degrees C to promote crystal growth. Scanning electron microscopy was used to investigate the microstructure of the glass-ceramics. X-ray diffraction was used to identify the crystalline phases in the glass-ceramics., Results: The ionomer glasses exhibited excellent flow ability. Crystallization could not be suppressed during heat-pressing. Very fine scale fluorapatite crystals were present in all of the samples after heat-pressing. Mullite and/or anorthite formed as a second crystal phase. On further heat-treatment of the samples, changes in crystal phases took place., Significance: Apatite was the main crystalline phase produced in the glass-ceramics; this factor is of clinical significance. In conclusion these glass-ceramics could be suitable for all-ceramic dental restorations.

Objectives: The processing route for two heat-pressed all-ceramic materials (Empress and OPC) is virtually identical. The purpose of this study was to determine the mechanical properties of both materials and determine if significant differences exist between them., Methods: X-ray powder diffraction of the ceramics before and after processing was carried out to identify the crystal phases present. The mechanical properties of both materials were tested. Specimens were tested for hardness, fracture toughness (indentation method) and flexural strength (biaxial method). The results were statistically evaluated and tested for differences using a Mann-Whitney test. Secondary electron imaging of both materials was carried out before and after processing., Results: X-ray powder diffraction revealed that OPC changes as a result of heat-pressing from being a complex mixture of crystalline oxides to a glass-ceramic. In contrast Empress is a glass-ceramic before and after processing. X-ray diffraction identified leucite as the main crystalline phase in both ceramics. The biaxial flexural strength of OPC was 153.6 (17.8) MPa and for Empress was 134.4 (11.5) MPa. The hardness of OPC was 7.28 (0.62) GPa and for Empress was 6.94 (0.79) GPa. Indentation fracture toughness of OPC was 1.36 (0.29) MPam0.5 and for Empress was 1.33 (0.08) MPam0.5. Secondary electron images show Empress to be the same before and after processing while OPC is clearly very different. Empress also appears to have a higher glass content compared with OPC., Significance: The results of X-ray diffraction show that Empress is pre-cerammed whilst OPC is not. Statistical analysis revealed that no significant difference exists between the two materials for any of the mechanical properties tested at a 95% (p < 0.05) confidence level. It was concluded that no difference exists between the two materials on completion of processing.

Purpose: The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys., Methods: Four female rhesus monkeys were dosed with lipid:DNA formulations via aerosol inhalation, where the DNA coded for the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein. Delivery of DNA was determined in lung samples by polymerase chain reaction (PCR) by qualitative and quantitative methods. Transgene specific messenger RNA was measured by reverse transcriptase PCR (RT-PCR) and protein expression and localization were evaluated by immunohistochemistry (IHC)., Results: Approximately four mg of DNA, complexed with cationic lipid 1.2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) and cholesterol were delivered to the lungs of animals by airjet nebulizer. Three days after dosing, tissue samples from the lung were collected and shown to have vector specific DNA, RNA and the presence of CFFR protein. Specifically, the hCFTR protein was distributed widely, although non-uniformly, throughout airway epithelium being located on the apical surface of epithelial cells. Importantly, no adverse clinical effects were observed and the lungs showed no histological abnormalities or signs of acute inflammation., Conclusions: This study shows that lipid:DNA formulations based on EDMPC and cholesterol can be administered to primates by nebulization resulting in measurable expression of the hCFTR protein. The absence of inflammation is also encouraging and such systems may have utility for delivery of genes to the lungs for the treatment of a variety of pulmonary diseases including cystic fibrosis.

Objective: Liposomes may be an appropriate transfection vehicle for transplanted hearts, avoiding the use of viruses in immunosuppressed hosts and allowing transfection of nondividing cells. To study whether liposome-mediated transfection could be accomplished during transplantation, we used a liposome-reporter gene system in a rabbit model of allograft cardiac transplantation., Methods: After aortic crossclamping, Stauffland donor hearts were injected with 10 ml Stanford cardioplegic solution; then a 1.3 to 2.0 mg/kg dose of chloramphenicol acetyl transferase in 1:1 deoxyribonucleic acid-liposome complexes was injected proximal to the aortic crossclamp for coronary artery perfusion. The hearts were transplanted into New Zealand White rabbit recipients in the heterotopic cervical position (n = 11 transplants). Recipients were sacrificed at 24 hours. Myocardial specimens (right and left ventricles) and vascular specimens (epicardial coronary artery, aortic root, and coronary sinus) from both the transplanted and native hearts were analyzed for chloramphenicol acetyl transferase protein by means of the enzymatic liquid scintillation assay (counts per minute per milligram of total protein)., Results: In the recipient, myocardial chloramphenicol acetyl transferase activity was significantly greater in treated donor hearts (mean 4.6 x 10(5) cpm/mg +/- 1.1 x 10(5) [standard error]) than in native hearts (mean 4.1 x 10(2) cpm/mg +/- 72 [standard error], p < 0.01, Mann-Whitney U test). In treated donor hearts, right and left ventricular specimens, as well as apical and basal myocardial specimens, were transfected equally. Chloramphenicol acetyl transferase activity in vascular specimens also indicated transfection (mean 5.4 x 10(5) cpm/mg +/- 2.5 x 10(5) [standard error]). Chloramphenicol acetyl transferase activity in the coronary sinus was comparable with that in the coronary arteries, which suggests that liposomes can transverse the coronary capillary beds., Conclusions: These findings demonstrate that ex vivo transfection of donor hearts with a liposome-reporter gene system results in significant in vivo expression of the transfected gene product after cardiac transplantation. Genetic alteration of myocardium and cardiac vasculature has potential clinical applications in the prevention of posttransplantation rejection, ischemia-reperfusion injury, and both transplant and nontransplant coronary artery disease.

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.

In this study the activation of the prohormone convertase mPC1 was determined. Expression and characterization of catalytic domain mutations (Ser382 to Ala or His208 to Ala) in the prohormone convertase mPC1, unequivocally demonstrated that pro-region cleavage proceeds by an autocatalytic mechanism. Furthermore, these results suggest that autoproteolysis may be the result of an intramolecular reaction, since proregion processing of the active-site mutant could not be complemented by the overexpression of active furin or PC1. Additionally coexpression of a cleavage-site mutant (Arg110-Ala) with the substrate prorelaxin further demonstrated that autoproteolysis is required for the full activity of PC1.

The conversion of human proinsulin to insulin occurs only in specialized cells which contain the appropriate processing enzymes. To allow proinsulin processing to occur in a wide variety of cell types, we engineered human proinsulin to be cleaved in the constitutive secretory pathway. Using site-directed mutagenesis, we have introduced furin consensus cleavage sequences (Arg-X-Lys-Arg) into the human proinsulin cDNA. These mutations allowed for efficient proteolytic maturation of human proinsulin to insulin within cells containing only a constitutive pathway of secretion. Additionally, a naturally occurring mutation (histidine B10 to aspartic acid) yields a form of human insulin which accumulates 10- to over 100-fold more mature insulin when compared to the mutants lacking this change. Engineering furin-specific cleavage sites into each junction of the human proinsulin cDNA results in the secretion of peptides that display the expected molecular weights for the A and B chains of insulin. The accumulation of mature, processed, human insulin was measured in the supernatants by radioimmunoassay, and the bioactivity of this molecule was measured by its ability to stimulate autophosphorylation of the insulin receptor. Our results suggest that any cell type might be engineered to produce mature, active, and stable insulin in the constitutive pathway of secretion.

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.

Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition. It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor. Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae. Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed. Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta. This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin. Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin. The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

Full-length tissue factor (263 rTF) and three truncated forms have been expressed in human kidney 293 cells; 1) 243 rTF, which lacks the cytoplasmic tail, is fully functional in the chromogenic assay and has a specific activity comparable with that of the full-length molecule, 263 rTF; 2) 219 rTF, which lacks both the transmembrane and cytoplasmic domains, is not functional; 3) the third variant, referred to as TF-PI, is a fusion protein containing the extracellular domain of TF (amino acids 1-219) fused to the last 37 amino acids of decay-accelerating factor which contain a signal for attachment of a phosphatidylinositol membrane anchor (PI). TF-PI is a membrane-bound protein expressed on the cell surface. The PI anchor restores TF activity lost when the transmembrane domain is deleted from the 219 rTF variant. The ability of the PI anchor to restore activity to 219 rTF clearly demonstrates that while the transmembrane domain is not required for TF activity, lipid association is required.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

Polymerase chain reaction analysis was used to identify aberrant splicing of the simian virus 40 small-t intron present in pRSVcat. We examined factors governing the selection and relative use of aberrant 5' splice sites derived from the chloramphenicol acetyltransferase-coding region. Our results indicated that transcripts from virtually any cDNA positioned upstream of the small-t intron could contain alternative 5' splice sites and therefore be subject to deletions within the protein-coding region.

Two expression vectors were constructed that differ only in the presence (+) or absence (-) of an intervening sequence (IVS) in their 5'-untranslated leaders. Transient transfection into four mammalian cell lines resulted in higher levels of the indicator protein (CAT) from the IVS(+) vector (6 to 50-fold). Cytoplasmic RNA concentrations in 293s and HeLa cell lines corresponded directly to resultant protein levels; measurements in 293s cells of transcription initiation and elongation, steady-state total nuclear RNA, and cytoplasmic RNA stability, were equivalent for the two vectors. Surprisingly, the amount of poly(A)+ nuclear RNA was greater from the IVS(+) vector. Since this difference matches the ratio seen with polyadenylated cytoplasmic RNA, our results imply that splicing is coupled to a polyadenylation/transport pathway.

We have examined the effect of adenovirus E1 proteins on expression from the immediate early (IE) region of the human cytomegalovirus (HCMV). The major immediate early promoter, responsive to trans-activation during the HCMV lifecycle, is also responsive to E1 a protein encoded by the 13 S message. E1a proteins inhibit SV40 expression through the mechanism of enhancer repression; however, the presence of E1a proteins did not inhibit expression of the IE region of HCMV. The ability of trans-activate the major IE promoter in the presence of a strong enhancer suggests adenovirus can activate transcription of HCMV upon coinfection. E1b proteins increased levels of steady state mRNA transcribed from the IE region. Increases in expression due to E1a and E1b proteins were additive. These results suggest that adenovirus early expression can activate quiescent HCMV sequences.

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Many viral genomes, including those of SV40 and MuLV, are not efficiently expressed in undifferentiated embryonal carcinoma (EC) cells but are expressed in differentiated derivatives. This regulation appears to be at the level of transcription. We have used DNA-mediated gene transfer to analyze the function of several viral promoters in EC cells. We show that the SV40 early promoter works efficiently in an enhancer-independent fashion following transfection into undifferentiated cells. Strikingly, the promoter in the LTR of MSV does not function in such cells; but when upstream sequences, including the enhancer, are deleted expression ensues. Replacement of the SV40 enhancer by that of MSV results in inactivation of the SV40 early promoter in these cells. We propose that the undifferentiated cells contain a trans-acting regulatory factor (or factors) that reduces transcription by interacting with viral enhancers.

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure.

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.

We have studied the effects of sodium butyrate on DNA-mediated gene transfer in an effort to investigate interrelationships between chromatin structure and expression of recombinant plasmids. Our results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels. First, the number of cells able to express foreign DNA increases from 10% to up to 40%. Second, there is an increase in enhancer-dependent transcription, approximately 30 fold in HeLa cells, involving the SV40 early promoter. Stable transformation efficiencies increase to 4% and 10% in HeLa S3 and monkey kidney CV-1 cells, respectively. Finally, expression of integrated recombinant plasmid genes is reinducible by a second treatment five weeks after initial exposure to this agent.

Human gamma-aminobutyric acid A (GABAA) receptor subunits were expressed transiently in cultured mammalian cells. This expression system allows the simultaneous characterization of ligand-gated ion channels by electrophysiology and by pharmacology. Thus, coexpression of the alpha and beta subunits of the GABAA receptor generated GABA-gated chloride channels and binding sites for GABAA receptor ligands. Channels consisting of only alpha or beta subunits could also be detected. These homomeric channels formed with reduced efficiencies compared to the heteromeric receptors. Both of these homomeric GABA-responsive channels were potentiated by barbiturate, indicating that sites for both ligand-gating and allosteric potentiation are present on receptors assembled from either subunit.

Tissue factor is a membrane protein that plays an essential role in the initiation of blood coagulation. When exposed to the circulation, tissue factor interacts with the serine protease factor VII, and the complex triggers fibrin clot formation by activating both factors IX and X of the coagulation cascade. This report describes the cloning and expression of the complementary DNA (cDNA) for human tissue factor. The cDNA encodes a protein of 263 amino acids preceded by a 32 amino acid signal peptide. The predicted protein sequence contains a potential hydrophobic membrane anchoring domain at its carboxy terminus, and bears no significant homology to any other known protein. Tissue factor mRNA of 2400 nucleotides was detected in adipose, adrenal, small intestine and a number of other tissues by Northern blot hybridization analysis. In order to confirm the identity of the cDNA, an expression vector containing the cloned cDNA was used to transfect cultured mammalian cells. These cells produced active tissue factor which was assayed using purified factors VII and X.

We have generalized the protocol of gene transfer, greatly increasing the variety of cells that can be used as recipients of foreign genes. Our approach has been to use a transient assay system that allows rapid screening of expression of foreign DNA. When the initial steps of gene transfer have been optimized with the transient system, these defined conditions are used to yield efficient stable transformation. We have seen that primate cells, including human cells, can be used in gene transfer experiments at levels sensitive enough to allow detection of single copy gene function. Recently we have also used this approach successfully with undifferentiated embryonic cells.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.

The efficiency of transfection and subsequent expression of recombinant DNA plasmids in monolayers of CV-1 monkey kidney cells was analyzed by immunoperoxidase and in situ hybridization with biotin-nucleotide-labeled DNA molecular probes. Two recombinant plasmids were used for transfection. Both contained the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) as the transcriptional promoter, but two different coding sequences were employed [bacterial chloramphenicol acetyltransferase (pRSVcat) and mouse casein alpha (pRSVcsn alpha)]. In our experiments up to 25% of the transfected cells were positive for pRSVcat expression by indirect immunoperoxidase assay with affinity-purified, biotinylated anti-goat gamma-globulin after exposure to goat anti-chloramphenicol acetyltransferase antibody. In duplicate cultures, where pRSVcat expression was monitored by in situ hybridization signal that was restricted to the cytoplasm in positive cells was identified as pRSVcat RNA by its sensitivity to alkali. Although transfection of CV-1 cells with pRSVcsn-alpha did not result in immunologically detectable alpha casein, greater than 14% of the cells possessed cytoplasmic RNA concentrations detectable by in situ hybridization. These observations provide comparative information on in situ hybridization and immunoperoxidase techniques. They further indicate that in situ hybridization can be used to evaluate the effectiveness of transfection with recombinant expression vectors.

The sequence poly(dT-dG).poly(dC-dA) (TG-element) is a ubiquitous component of eucaryotic genomes and has the potential to adopt a left-handed DNA conformation (Z-DNA). In this report, we have tested the hypothesis that the TG-element can modulate gene expression. Human genomic DNA fragments (1 to 1.5 kilobases) containing a (dT-dG)n.(dC-dA)n tract (30, 40, or 50 base pairs) or chemically synthesized (dT-dG)n.(dC-dA)n fragments (50 to 130 base pairs) were inserted in the pSV2-cat (simian virus 40 enhancer plus) or pA10-cat (enhancer minus) expression vector plasmid. These constructs were transfected into CV-1 cells or HeLa cells, and their transcription was monitored by assaying chloramphenicol acetyltransferase activity. The results showed that pSV2-cat with the TG-element and pA10-cat with the TG-element synthesized more chloramphenicol acetyltransferase activity (2 to 10 times, depending on the location of the TG-element) than did parental pSV2-cat and pA10-cat DNAs, respectively. Furthermore, the TG-element appeared to have characteristics similar to those of viral enhancers: (i) the TG-element enhanced transcription from a distance, (ii) its closer location to the promoter was more effective, and (iii) its orientation was not crucial. However, its enhancer-like activity was much weaker than that of the simian virus 40 enhancer, and, unlike many viral enhancers, it was equally active in monkey and in human cells. These results suggest that the TG-element may influence the expression of cellular genes.

Hybrid prokaryotic/eukaryotic expression vectors have been used to introduce Drosophila histone genes into CV-1 African green monkey tissue culture cells. Transfection of CV-1 cells with Drosophila genes under the control of insect DNA promoter sequences results in low level expression of histone genes. On the other hand, when the Drosophila H2a gene is juxtaposed downstream from the long terminal repeat sequence of Rous sarcoma virus (RSV) expression of the insect gene is considerably more efficient; both 3' polyadenylated insect histone messenger RNA and putative Drosophila H2a histone protein can be readily detected in the transduced cells. Using this RSV/H2a vector, we have been able to demonstrate the presence of Drosophila H2a histone in monomer nucleosome preparations isolated from transfected CV-1 cells. These results suggest the feasibility of 'remodeling' cellular chromatin in vivo in precisely defined ways. The techniques described may be generally applicable to other genes coding for chromosomal proteins.