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Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

A commonly used approach to control recombinant protein production in Escherichia coli utilizes the lambda pL promoter-operator and the lambda repressor. Inactivation of the lambda repressor function allows transcription to proceed. However, induction of the RecA-mediated cleavage of lambda repressor by the addition of nalidixic acid or inactivation of a temperature-sensitive lambda repressor by growth at the nonpermissive temperature can have detrimental effects on protein production. This paper describes the use of an alkaline shift in the pH of the growth medium that allows expression of genes from the pL promoter in a RecA-independent manner. This procedure results in high-level production of recombinant protein. The pH shift is performed in the stationary phase of cell growth, using culture volumes ranging from 1-1000 ml. This method can result in the production of over 15-fold more active protein than when using a temperature shift.

Adenovirus vectors have been shown to be highly effective as vaccine platforms capable of inducing both humoral and cell mediated immune (CMI) responses. An Ad serotype 5 vector containing unique deletions in the E2b region (Ad5 [E1-, E2b-]) has been reported to have several advantages over conventional Adenovirus serotype 5 (Ad5) vectors deleted in only the E1 region (Ad5 [E1-]), including increased carrying capacity and diminished viral late gene expression. Here, we evaluated a novel Ad5 [E1-, E2b-] vector utilizing the E.C7 cell line for viral packaging. Its' effectiveness as a potential vaccine platform as compared to the currently utilized Ad5 [E1-]-based platform was assessed in both Ad5 naive and Ad5 immune mice. We employed the HIV-1 Gag gene as the antigenic transgene expressed by the novel vector. Cellular expression of the Gag was confirmed by Western Blot analysis. Dose response studies using three intradermal immunizations of 10(7) to 10(10) virus particles (VP) of each construct revealed that immunization with 10(10)VP resulted in the maximum immunological response. Multiple immunizations of Ad naive BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in higher ELISpot CMI responses as compared to mice immunized with an Ad5 [E1-]-gag vaccine. More importantly, multiple immunizations of Ad5 immune BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in significant increases in ELISpot CMI responses when compared to Ad5 immune mice vaccinated with an Ad5 [E1-]-gag vector. Preliminary studies in three Ad5 immune non-human primates (NHP) demonstrated that vaccination with Ad5 [E1-, E2b-]-gag-induced elevated levels of interferon-gamma and IL-2 secreting lymphocytes as assessed by ELISpot assays. These studies indicate that the novel Ad5 [E1-, E2b-] viral vector can be utilized as a potential vaccine platform to induce elevated CMI responses as compared to current generation Ad5 [E1-] viral vectors even in the presence of pre-existing Ad5 immunity.

A series of mutations comprising single and multiple substitutions, deletions, and extensions within the carboxy-terminal domain of the bacteriophage lambda Cro repressor have been constructed. These mutations generally affect the affinity of repressor for specific and nonspecific DNA. Additionally, substitution of the carboxy-terminal alanine with several amino acids capable of hydrogen-bonding interactions leads to improved specific binding affinities. A mutation is also described whereby cysteine links the two Cro monomers by a disulfide bond. As a consequence, a significant improvement in nonspecific binding and a concomitant reduction in specific binding are observed with this mutant. These results provide evidence that the carboxy terminus of Cro repressor is an important DNA binding domain and that a flexible connection between the two repressor monomers is a critical factor in modulating the affinity of wild-type repressor for DNA.

Abstract —During their 7- to 9-day lifespan in the circulation, platelets perform an ill-defined baseline function that maintains the integrity of the vasculature. In thrombocytopenic states, there is an increase in vascular permeability and fragility, which is presumably due to absence of this platelet function. In sharp contrast, biochemical or physical injury in the coronary, carotid, or peripheral arteries induces platelet activation and platelet recruitment, which can progress to thrombotic vascular occlusion. Because there is 1 death every 33 seconds from vascular occlusion in the United States, this problem has critical public health implications. In this review, we describe the characterization of a novel potential antithrombotic agent with a unique mode of action—biochemical “deletion” of ADP from an activated platelet releasate, which thereby inhibits platelet recruitment and further activation.

Proteins without readily available three-dimensional structural data present a difficult problem in the exploration of structure/function relationships. Saturation mutagenesis using contaminated oligonucleotides can identify potentially interesting regions of such a protein. This technique, in which synthesized oligonucleotides contain low-level base substitutions, allows random mutations to be placed throughout a gene sequence. Using double-stranded cassettes, a region of the human interleukin-1 alpha gene has been altered using such mutagenic oligonucleotides. However, instead of contaminating both strands of the gene sequence at the same level, each strand of the insert was contaminated at a different level. Several recombinants were sequenced and the effects of the mutations on the activity of the proteins were examined. Contaminating the two oligonucleotides at different levels produced a significantly different distribution of nucleotide changes from that seen if both strands were contaminated at the same level. The observed distribution followed the average of the distributions for each of the two contamination levels. This resulted in roughly equal frequencies of 1 to 5 nucleotide changes per clone with very few clones containing the wild-type nucleotide sequence. This helped overcome the redundancy in the genetic code, resulting in a high frequency of amino acid changes, and allowed changes at every amino acid to be sampled in a small number of mutants. This procedure can allow a gene sequence to be screened rapidly by removing most wild-type sequences from analysis while making sure that there are many amino acid changes in the resultant mutants.

A region upstream from the origin of replication in Co1E1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other Co1E1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Publisher Summary This chapter discusses the method for the synthesis of a specific sequence of DNA using restriction enzymes. The restriction enzyme MboII recognizes the DNA sequence GAAGA and cleaves the DNA eight bases downstream from this site, leaving a single 3' protruding base. There are no apparent sequence requirements in the stretch of DNA between the recognition sequence and the point of cleavage. Using this property, a segment of DNA from one fragment can be transferred to another fragment. The scheme can be demonstrated by the formation of a restriction enzyme recognition site from two MboII cleavage products. The extension of the basic scheme would allow the production of more generalized fragments useful in constructing gene sequences.

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3′ phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.