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The demand of therapeutic protein production from mammalian cells has expanded greatly since the first biologic was approved in 1982. It remains a major challenge to exploit the exocytic pathway and increase cell viability during the production process. Hepatitis B virus X protein (HBx) is a multifunctional viral transcription activator that regulates a variety of cellular events including transcription, cell cycle and proliferation, survival, and apoptosis. As such it may address some of the current production challenges. In this study we demonstrate that HBx can enhance protein production during transient transfection and in stable cell lines. XBP1s is a potent transcription factor and has been demonstrated to enlarge the ER secretion pathway and increase protein production. We explored the possibility of combinational engineering of HBx with XBP1s in BHK21 cells. Our data revealed that combinational engineering of HBx with XBP1s further enhances protein production compared with HBx or XBP1s alone., (2009 Wiley Periodicals, Inc.)

Widespread adaptation of small molecule-regulated expression systems requires the development of selective inducer molecules that do not have any significant side effects on the endogenous receptors from which the regulated expression system is derived. Here we report the identification and in vitro validation of a novel inducer-receptor pair for the single-plasmid regulated expression system termed pBRES, which contains the ligand-binding domain from the human progesterone receptor (hPR). A small molecule inducer, BLX-913, has been identified as having a 30-fold lower IC(50) for the human progesterone receptor than mifepristone (MFP), the previously best characterized inducer for pBRES. Using modeling-guided protein engineering, compensatory mutations were installed at positions W755 and V729 (hPR numbering) in the ligand-binding pocket of the pBRES regulator protein (pBRES RP) to accommodate the new inducer and allow induction of transgene expression to levels previously seen with MFP. The improved inducer-pBRES RP complex was validated in vitro by monitoring the induction of luciferase, murine secreted alkaline phosphatase, and human interferon beta transgenes in mouse skeletal muscle cells. The engineered pBRES demonstrated low levels of transgene expression in the absence, and high expression levels in the presence, of the new BLX-913 inducer. Findings presented here allow induction of the pBRES-regulated gene expression system by a compound with markedly lower anti-hPR activity than MFP, the previously best characterized inducer.

Background: Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term "Directed Evolution" for creating highly potent oncolytic viruses., Methodology/principal Findings: Taking the "Directed Evolution" approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2-3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent., Conclusions/significance: Using the "Directed Evolution" methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies.

This study illustrates the application of asymmetrical flow field-flow fractionation (AF4) and light scattering analysis during the development of a gene delivery vehicle based on virus-like particles (VLPs) derived from the human polyoma JC virus. The analytical system was created by connecting an AF4 apparatus to the following detectors: diode array, fluorescence, multiangle light scattering, dynamic light scattering, and refractometer. From a single analysis, the molar mass, root mean square and hydrodynamic radii, composition, and purity of the sample could be determined. The VLPs were purified from baculovirus-infected Sf158 insect cells overexpressing the recombinant VP1 protein using weak anion exchange chromatography. The VLPs were dissociated to VP1 pentamers, and the contaminating DNA and proteins were removed using strong anion exchange chromatography. The gene delivery vehicle was created by reassembling the VP1 pentamers in the presence of the desired DNA. The newly formed VLPs encapsulated the DNA and were shown to be capable of delivering the gene of interest to target cells where it was translated into protein. This paper describes the scalable process that was derived to produce the VLPs and demonstrates how the AF4-based analytical characterization was indispensable during the development process.

A single plasmid regulated expression vector based upon a mifepristone-inducible two plasmid system, termed pBRES, has been constructed and tested in mice using murine interferon-b (mIFNb) as the transgene. The expression of mIFNb in the circulation was followed by measuring the systemic induction of IP-10, a validated biomarker for mIFNb in mice. Long-term, inducible expression of mIFNb was demonstrated following a single intramuscular (i.m.) injection of the pBRES mIFNb plasmid vector into the hind limb of mice. Induction of mIFNb expression was achieved by administration of the small molecule inducer, mifepristone (MFP). Plasmid DNA and mIFNb mRNA levels in the injected muscles correlated with mIFNb expression as monitored by IP-10 over a 3-month time period. Renewable transgene expression was achieved following repeat administration of the plasmid at 3 months following the first plasmid injection. A dose-dependent increase in expression was demonstrated by varying the amount of injected plasmid or the amount of the inducer administered to the mice. Finally, the pBRES plasmid expressing mIFNb under control of the inducer, MFP, was shown to be efficacious in a murine model of experimental allergic encephalomyelitis, supporting the feasibility of gene-based therapeutic approaches for treating diseases such as multiple sclerosis.

Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 x 10(9) and 5 x 10(10) virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-beta (AAV1-hIFNbeta). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 x 10(10) vp of AAV1-IFNbeta were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNbeta expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.

We have developed a one-plasmid regulated gene expression system, pBRES, based on a mifepristone (MFP)-inducible two-plasmid system. The various expression elements of the pBRES system (promoters, 5' and 3' untranslated regions (UTRs), introns, target gene, and polyA sequences) are bounded by restriction enzyme sites so that each module can be conveniently replaced by alternate DNA elements in order to tailor the system for particular tissues, organs, or conditions. There are four possible orientations of the two expression units relative to each other, and insertion of a variety of expression elements and target genes into the four different orientations revealed orientation- and gene-dependent effects on induced and uninduced levels of gene expression. Induced target gene expression from the pBRES system was shown to be comparable to the two-plasmid system and higher than the expression from the cytomegalovirus (CMV) promoter in vivo, while maintaining low uninduced levels of expression. Finally, a pBRES expression cassette was transferred to an adeno-associated virus (AAV) vector and shown to be capable of regulated gene expression in vivo for nearly 1 year.

Recombinant interferon-beta (IFN-beta) protein is used successfully for the treatment of multiple sclerosis (MS). Gene therapy might be an alternative approach to overcome drawbacks occurring with IFN-beta protein therapy. A critical issue in developing a new approach is detection of biologically active IFN-beta in preclinical models. The goal of the present study was to determine if Mx1 and IP-10, which are known to be activated after IFN-beta treatment in humans, can be used as biomarkers in mice. In three in vivo experiments, the correlation between different methods of murine IFN-beta (MuIFN-beta) delivery and biomarker induction was studied: (1) bolus protein delivery by intravenous (i.v.) or intramuscular (i.m.) injection, (2) gene-based delivery of IFN- beta by i.m. injection of plasmid DNA, followed by electroporation, and (3) gene-based delivery of IFN-beta by i.m. injection of adenovirus-associated type 1 (AAV1). Short-term induction of Mx1 mRNA and IP-10 was observed after treatment with bolus MuIFN-beta protein. Long-term induction of both biomarkers was observed after IFN-beta plasmid DNA delivery or when AAV1 was used as the vector. The experiments demonstrate that gene-based delivery provides sustained levels of IFN-beta compared with bolus protein injection and that Mx1 RNA and IP-10 can be used to monitor biologically active circulating plasma MuIFN-beta protein in mice.

Experimental allergic encephalomyelitis (EAE) is a model of central nervous system (CNS) inflammation that follows immunization with certain CNS antigens. The course and clinical manifestations of EAE are similar to those of multiple sclerosis (MS) in humans; therefore, EAE has become an accepted animal model to study MS. The purpose of this study was to demonstrate that systemic expression of murine interferon-beta (IFN-beta) (MuIFN-beta), following intramuscular (i.m.) delivery of plasmid DNA encoding MuIFN-beta to the hind limb of mice, is effective in reducing the clinical manifestations of disease in a model of EAE. The results of the study demonstrate that gene-based delivery of MuIFN-beta caused significantly decreased clinical scores compared with delivery of the null vector. A single injection of the MuIFN-beta plasmid was as effective in reducing the severity of the disease as an every other day injection of MuIFN-beta protein.

Background: Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain., Methods: Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing., Results: The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7., Conclusions: Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors., (Copyright 2004 John Wiley & Sons, Ltd.)

The EGF domain of heregulin has all the receptor binding characteristics of full-length heregulin and has strong homology to the ligands for erbB-1. Despite this, it does not bind erbB-1 but instead binds erbB-3 and erbB-4. The sequence similarity between HRG and the erbB-1 ligands suggest that a few residues are responsible for receptor binding specificity. To determine the sequences involved in receptor binding, we performed homologue scanning mutagenesis on the EGF domain of HRGalpha using sequences of TGFalpha or EGF. We found three sets of mutations in the N-terminal subdomain that were responsible for receptor binding specificity. Mutations in the C-terminal subdomain affected the binding affinity, but did appear to confer any specificity., (Copyright 1998 Academic Press.)

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.

The type I interferon (IFN) receptor complex is assumed to be composed of multiple protein subunits. Recently, two proteins have been identified as potential receptor components, both of which share a high degree of structural homology with the immunoglobulin superfamily. One of these proteins, referred to as the human interferon alpha receptor (IFNAR), has been shown to be involved in interferon signal transduction, but it does not bind IFN with high affinity. A second putative receptor protein, named FLP40, has been cloned from human Daudi cells. Transfection of FLP40 into murine NIH 3T3 cells does not result in high affinity IFN binding. In this study, we demonstrate that when expressed in murine L929 cells neither IFNAR nor FLP40 by themselves are capable of binding human IFN-alpha 8. Co-expression of IFNAR and FLP40 results in cells capable of binding IFN-alpha 8 and IFN-alpha 2. Scatchard analysis of binding demonstrated the presence of high (KD 350 pM) and low (KD 4.0 nM) affinity binding sites. Binding of radiolabeled IFN-alpha 8 can be competed with either unlabeled IFN-alpha 8 or a recombinant form of human interferon beta, IFN-beta 1b, but not with IFN-gamma. Ligand binding of IFN-alpha 8 can be inhibited by antibodies directed against IFNAR providing further support for a role for this protein in the formation of a ligand binding site. This is the first demonstration indicating that two previously identified IFN receptor proteins, which individually do not bind type I IFN with high affinity, cooperate in the formation of a type I IFN receptor ligand binding complex.

HER2, the erbB-2/neu proto-oncogene product, is a 185-kDa transmembrane glycoprotein related to the epidermal growth factor receptor. Overexpression of HER2 was reported in several human adenocarcinomas, including mammary and ovarian carcinomas. A family of glycoproteins, the heregulin/neu differentiation factors, was characterized and implicated as the ligands for HER2. Recently, it has been shown that HER2 alone is not sufficient to reconstitute high affinity heregulin receptors and that HER3 or HER4 may be the required components of the heregulin receptors on mammary carcinoma cells (Sliwkowski, M.X., Schaefer, G., Akita, R.W., Lofgren, J.A., Fitzpatrick, V.D., Nuijens, A., Fendly, B.M., Cerione, R.A., Vandlen, R.L., and Carraway, K.L., III (1994) J. Biol. Chem. 269, 14661-14665; Plowman, G.D., Green, J.M., Culouscou, J.-M., Carlton, G.W., Rothwell, V.M., and Buckley, W. (1993) Nature 366, 473-475). Using the Cytosensor to measure the extracellular acidification rate, we have examined the effects of recombinant human heregulin-alpha on three mammary carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7), an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1 and 293-EBNA cells stably transfected with HER2. By reverse transcription polymerase chain reaction and Western blotting, we found that the breast cells also express HER3 and that the ovarian line co-expresses the HER4 message. A dramatic increase in the acidification rate was observed for the mammary carcinoma cells co-expressing high levels of HER2 and HER3. In contrast, the ovarian cells expressing high levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells expressing HER2 alone were not responsive to heregulin. When these same transfected cells were exposed to monoclonal anti-HER2 antibody followed by anti-IgG to cause aggregation of the HER2 molecules, an increase in the acidification rate was observed, indicating coupling of transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced acidification responses. These data, together with the previously reported high affinity heregulin binding and activation of tyrosine phosphorylation in HER2 and HER3 co-transfected cells support the role of HER2 and HER3 as components of the heregulin receptor in breast cells.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.

The interaction of transforming growth factor alpha (TGF-alpha) with the complete extracellular domain of the epidermal growth factor receptor (EGFR-ED) was examined by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR resonances of the methyl groups of TGF-alpha were used as probes of the interaction of TGF-alpha with the EGF receptor to determine the binding kinetics and the differential mobility within the bound TGF-alpha. The methyl resonances were studied because there are 14 methyl containing residues well dispersed throughout the structure of TGF-alpha and the relaxation properties of methyl groups are well understood. Changes in the longitudinal and transverse 1H NMR relaxation rates of the methyl resonances of TGF-alpha caused by binding to the 85-kDa EGFR-ED were studied. From these measurements it was determined that the interaction was in the NMR fast exchange limit. A binding mechanism to rationalize the different rates determined by NMR and surface plasmon resonance techniques [Zhou, M., et al. (1993) Biochemistry 32, 8193-8198] is proposed. The transverse relaxation rate (R2) enhancements of the various methyl resonances displayed a regional dependence within the bound TGF-alpha molecule. Resonances from the C-terminus of TGF-alpha, which were flexible in the unbound molecule, revealed dramatic increases in their R2 upon binding to the EGFR-ED along with resonances from the interior of TGF-alpha. However, upon binding, the R2 enhancements of the methyl resonances from the N-terminus of TGF-alpha, which were also flexible in the unbound TGF-alpha, were slight; indicating a retention of mobility of this region for bound TGF-alpha. The implications of these data with respect to the mechanism of receptor activation and the design of antagonists are discussed.

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. & Van Montagu, M. Proc. Natl. Acad. Sci. USA (1990) 87, 4859-4863]. We report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5' and 3' ends. Gene-specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes.

To define the role of TGF alpha in normal tissue function and in pathogenesis, transgenic mice have been generated bearing a fusion gene consisting of the mouse metallothionein 1 promoter and a human TGF alpha cDNA. In these mice, human TGF alpha RNA and protein are abundant in many tissues and TGF alpha is detectable in blood and urine. The effects of TGF alpha overproduction in transgenic mice are pleiotropic and tissue specific. The liver frequently contains multifocal, well-differentiated hepatocellular carcinomas that express enhanced levels of human TGF alpha RNA. The mammary gland exhibits impeded morphogenetic penetration of epithelial duct cells into the stromal fat pad. The pancreas shows progressive interstitial fibrosis and a florid acinoductular metaplasia, during which acinar cells appear to degranulate, dedifferentiate, and assume characteristics of intercalated or centroacinar duct cells. TGF alpha therefore plays an important role in cellular proliferation, organogenesis, and neoplastic transformation.

The amino acid sequence of human lymphotoxin derived from a 1788 lymphoblastoid cell line was determined. Peptide fragments obtained by trypsin, lysine-C peptidase, cyanogen bromide, and acetic acid cleavage of the intact protein were purified by reverse-phase high performance liquid chromatography and analyzed by amino acid composition and by automated Edman degradation. The protein is 171 amino acids long with a molecular weight of 18,664. It contains one asparagine-linked glycosylation site and lacks cysteine. The salient features of the amino acid sequence of lymphotoxin are described.

Pyruvate kinase was purified from cat and trout muscle. The enzymes had similar amino acid compositions and subunit molecular weights. In contrast to the mammalian enzyme, the trout muscle pyruvate kinase was activated by fructose 1,6-bisphosphate. However, unlike the L-type pyruvate kinase from mammalian liver it was not phosphorylated by cyclic-AMP-dependent protein kinase. The purified enzyme from cat muscle was carboxymethylated with iodo[2-14C]acetic acid under conditions that led to the preferential labelling of one especially reactive thiol group. The labelled enzyme was cleaved with CNBr, and the radioactive fragment purified. Amino acid sequence analysis of the reactive-thiol-containing fragment from cat muscle pyruvate kinase showed it had the following sequence: Ile-Gly-Arg-[14C]CmCys-Asn-Arg-Ala-Gly-Lys-Pro-Val-Ile-CmCys-Ala-Thr-Gln- Hse. The corresponding peptide from trout pyruvate kinase had only one difference in its amino acid composition and the sequence around the reactive thiol was identical.

We have computed the observed distribution of amino acid pairs n, n+1 up to n, n+10 within 100 unrelated protein sequences containing a total of 14,034 amino acids. The expected distribution of the same amino acid pairs was calculated assuming that the amino acids within each sequence are arranged in random order. Statistical analysis of the data shows that amino acids in the n, n+1 and n, n+3 relationship deviate from random expectation at the 1% level of significance. These relationships are consistent with known secondary structures; however, there are no amino acid pairs which obviously contribute to the non-random result.

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.

The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.

We have compiled the dipeptide frequencies in 100 known protein sequences. We suggest that dipeptides which occur with low frequencies can be used to locate proteins where partial gene duplication may have taken place. The 48 residue sequence of posterior pituitary peptide contains two Cys Trp pairs. The adjacent portions of the sequence are compatible with a partial gene duplication in the evolutionary history of posterior pituitary peptide.

A 2760-base pair DNA segment of the Pseudomonas aeruginosa strain PA103 chromosome encoding the exotoxin A (ETA) structural gene has been cloned in Escherichia coli and the nucleotide sequence has been determined. Analysis of the 5'- and 3'-flanking regions indicate that ETA is translated from a monocistronic message. Comparison of the deduced NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein indicates that ETA is made as a 638 amino acid precursor from which a highly hydrophobic leader peptide of 25 amino acids is removed during the secretion process. Data are presented that indicate a COOH-terminal location of the ADP-ribosylation activity of the molecule. Expression of the ETA coding sequence in E. coli under control of the E. coli trp promoter, but not the ETA promoter, results in the production of enzymatically and immunologically active protein. However, most of this material appears to be neither processed nor secreted. Comparison of the ETA amino acid and nucleotide sequences to those of diphtheria toxin revealed no significant homologies, indicating that these functionally similar toxins evolved from different genes.

Murine peritoneal exudate cells (PEC) treated with murine recombinant interferon-gamma (IFN-gamma) (greater than 99% estimated purity), or concanavalin A-stimulated spleen cell supernatants developed tumoricidal properties (macrophage activation factor [MAF] activity). MAF activity was found to occur with treatments of 10 U/ml IFN-gamma, and at levels as low as 1 U/ml IFN-gamma if a second signal (5 ng/ml endotoxin) was present in the MAF assay. Endotoxin (lipopolysaccharide [LPS]) alone at these levels failed to induce MAF; induction of MAF was observed at 1,000-fold greater levels. The ability of IFN-gamma to stimulate murine PEC was species specific. Various sources of materials that displayed MAF activity, including supernatants from interleukin 2-dependent cloned cytotoxic murine T lymphocyte lines that did not display detectable antiviral activity, were neutralized by antibody raised and affinity purified against recombinant IFN-gamma. Thus, IFN-gamma, although never detectable by antiviral assays, appears to be present in many lymphokine preparations and has potent macrophage activation capability.

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8-12 residue peptide from the C-terminus.

A unique, efficient, and inexpensive system has been designed and built for the automatic conversion of anilinothiazolinone derivatives extracted from a Beckman spinning-cup sequencer with subsequent on-line high-pressure liquid chromatography separation of the phenylthiohydantoin derivatives. The Auto Converter-Auto Sampler system is controlled by a tape programmer or microprocessor and operates by transfer of the sample from the conversion vial into an HPLC injection loop by nitrogen pressure. Incorporation of a minor programming change on the sequencer allows the introduction of nitrogen vapor into the spinning cup during phenylisothiocyanate coupling. These modifications have resulted in a completely automated subnanomole protein sequencer.

The effects of bacteria-produced human interferons (HuIFN) alpha, beta, and gamma on in vitro immune functions of human peripheral blood mononuclear cells (PBMC) were studied. Proliferative response to phytohemagglutinin was significantly inhibited by the addition of HuIFN-alpha 2 or HuIFN-beta at 10, 100, or 1000 U/ml. In contrast, HuIFN-gamma showed suppressive activities only when added at 1000 U/ml. HuIFN-alpha 2 or HuIFN-beta caused significant inhibition of human mixed-lymphocyte reaction (MLR) as measured by [3H]thymidine incorporation. Similar inhibition was caused by HuIFN-gamma when it was added only at very low concentrations (1 U/ml); 10, 100, or 1000 U/ml resulted in no or only a modest increase in MLR. All three interferons exhibited dose-related effects on PWM-induced immunoglobulin synthesis in cultures of PBMC. These data demonstrate that purified interferons produced by recombinant DNA technology can significantly alter in vitro immune functions and that HuIFN-gamma has properties which are different from those of HuIFN-alpha 2 or HuIFN-beta.

The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5.

Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte beta-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membrane-spanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The beta-adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins.

The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.

Human lymphotoxin was purified to homogeneity from a serum-free tissue culture supernatant of a lymphoblastoid 1788 cell line. The purification scheme consisted of DEAE-cellulose chromatography, preparative isoelectric focusing, lentil lectin-Sepharose chromatography, and preparative polyacrylamide gel electrophoresis. The purified glycoprotein was homogeneous by the criteria of high pressure liquid chromatography and polyacrylamide gel electrophoresis run under both nondenaturing and denaturing conditions. The specific activity of the purified lymphotoxin is approximately 40 X 10(6) units/mg. The protein has an apparent molecular weight of approximately 20,000, and RF of 0.33 on 7.5% polyacrylamide gels at pH 8.8 and an isoelectric point of 5.8. A tryptic digest of the purified native material produced two major fragments of approximately 15,000 and 5,000 Da. The amino acid compositions of the intact molecule and of the tryptic fragments are presented.

Estrogens stimulate the synthesis of specific secretory proteins in the rat uterus. Here we show that two of these, polypeptides of relative molecular weight 110,000 (110K) and 74,000 (74K), are structurally related to C3, the third component of complement, a glycoprotein that plays a central role in regulating complement-mediated inflammatory and immune responses. The similarities were based on the observations that (1) NH2-terminal amino acid sequence of the 74K polypeptide showed sequence homology with the beta chain of mouse C3, (2) comparison of the electrophoretic mobilities of the 110K and 74K polypeptides in the presence and absence of reducing agents revealed that they were disulfide-linked subunits of a protein of Mr approximately 180,000, (3) the native protein was immunoreactive with antibodies specific for rat C3, and (4) both polypeptides were immunoprecipitated with antibodies to rat C3.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.

The signal recognition particle (SRP) receptor is an integral membrane protein of the endoplasmic reticulum which, in conjunction with SRP, ensures the correct targeting of nascent secretory proteins to this membrane system. From the complementary DNA sequence we have deduced the complete primary structure of the SRP receptor and established that its amino-terminal region is anchored in the membrane. The anchor fragment and the cytoplasmic fragment contribute jointly to a functionally important region which is highly charged and may function in nucleic acid binding.

Recombinant DNA methodology has allowed the production of human fibroblast interferon (IFN-beta) from Escherichia coli and this material, in highly purified form, has been shown to reduce viraemia and mortality in encephalomyocarditis (EMC) virus-infected squirrel monkeys. These effects are dose related: six treatments over 4 days at 10(6) U/kg and 3 x 10(3) U/kg have comparable efficacy, whereas treatments at 10(3) U/kg are ineffective. The recombinant DNA-derived IFN-beta appears to be as effective as natural fibroblast cell-derived IFN-beta and both materials are effective by the intramuscular or intravenous routes. Thus, even though previous studies have shown that low circulating concentrations of IFN-beta are observed after intramuscular injections, the present data indicate that this slow release from the muscle can still confer protection.