Economic analysis of CDC's culture- and smear-based tuberculosis instructions for Filipino immigrants.

SETTING: In 2007, the US Centers for Disease Control and Prevention (CDC) revised its tuberculosis (TB) technical instructions for panel physicians who administer mandatory medical examinations among US-bound immigrants. Many US-bound immigrants come from the Philippines, a high TB prevalence country. OBJECTIVE: To quantify economic and health impacts of smear- vs. culture-based TB screening. DESIGN: Decision tree modeling was used to compare three Filipino screening programs: 1) no screening, 2) smear-based screening, and 3) culture-based screening. The model incorporated pre-departure TB screening results from Filipino panel physicians and CDC databases with post-arrival follow-up outcomes. Costs (2013 $US) were examined from societal, immigrant, US Public Health Department and hospitalization perspectives. RESULTS: With no screening, an annual cohort of 35 722 Filipino immigrants would include an estimated 450 TB patients with 264 hospitalizations, at a societal cost of US$9.90 million. Culture-based vs. smear-based screening would result in fewer imported cases (80.9 vs. 310.5), hospitalizations (19.7 vs. 68.1), and treatment costs (US$1.57 million vs. US$4.28 million). Societal screening costs, including US follow-up, were greater for culture-based screening (US$5.98 million) than for smear-based screening (US$3.38 million). Culture-based screening requirements increased immigrant costs by 61% (US$1.7 million), but reduced costs for the US Public Health Department (22%, US$750 000) and of hospitalization (70%, US$1 020 000). CONCLUSION: Culture-based screening reduced imported TB and US costs among Filipino immigrants.

State and Local Perspective on Implementation of the Centers for Disease Control and Prevention Dog Confinement Agreement.

The Centers for Disease Control and Prevention (CDC) works in conjunction with state, territorial, local and tribal agencies (STLTAs) to prevent the transmission of infectious agents. Issuance of confinement agreements using CDC Form 75.37 'Notice to Owners and Importers of Dogs' to importers of dogs that are not vaccinated or incompletely vaccinated against rabies is part of the agency's regulatory programme to prevent the entry of dogs infected with rabies. Although this is a regulatory programme that depends heavily on partnerships between CDC and STLTAs, CDC had never formally evaluated the acceptability of the confinement agreement process with these partners. Thus, a short survey of nine STLTAs was conducted to evaluate whether these partners have enough personnel and resources to implement the regulation and their general opinions of the confinement agreement process. The results illustrate that CDC partners are dissatisfied to some extent with the process, and there are multiple issues limiting their success in enforcing the regulation.

T-cell specific avian TdT: characterization of the cDNA and recombinant enzyme.

A cDNA clone coding for avian terminal deoxynucleotidyl transferase (TdT) has been isolated and sequenced. The size of this cDNA was 2545 bp with an open reading frame of 1521 bp and a predicted translation product of 58 kDa. Comparison of this TdT sequence with other known TdT sequences has revealed a very high degree of homology at both the DNA and predicted amino acid levels. The chicken TdT cDNA was expressed in a bacterial system and the protein was purified by affinity chromatography. The purified recombinant enzyme, with a specific activity of approximately 1700 U/mg protein, was significantly less active than TdTs from mammalian species. This finding correlates with the observation that TdT isolated from avian thymus has lower activity than that isolated from any mammalian thymus source. Northern blot hybridization analyses and reverse transcription PCR of RNA preparations were carried out with the chicken cDNA. The data generated from these experiments revealed that the TdT RNA was only expressed in the thymus and not in the bone marrow or the bursa of Fabricius during pre- and post hatching chicken development. These data suggest that while TdT is probably involved in N region addition in chicken T-cell receptor genes, it is unlikely to play a role in diversification of immunoglobulin genes.

Initiation of transcription at the human terminal deoxynucleotidyl transferase gene promoter: a novel role for the TATA binding protein.

Control of initiation of transcription of the human terminal deoxynucleotidyl transferase (TdT) gene was investigated by using an in vitro transcription assay. The precise contribution of discrete basal promoter elements to transcription initiation was determined by testing deletion and substitution mutations. The primary element, contained within the region spanning -34 to -14 bp relative to the transcription start site, accounted for 80% of basal promoter activity. TdT promoter activity required the sequence ACCCT at -24 to -20 bp since a dramatic decrease in transcription initiation was observed after mutation of this sequence, whereas mutation of the adjacent sequence from -32 to -25 bp did not alter promoter activity. The secondary element contained sequences surrounding the transcription start site and had 20% of promoter activity. Deletion of both elements completely abolished transcription initiation. Initiator characteristics of the secondary element were revealed by using the in vitro assay: promoter sequences at the transcription start site were sufficient to direct accurate initiation at a single site. Mutation of the sequence GGGTG spanning the transcription start site resulted in loss of transcription initiation. Both the primary and secondary elements were nonhomologous to corresponding regions from the mouse TdT gene promoter. While the human basal promoter functioned in the absence of TATA consensus sequences or GC-rich SP1 binding sites, it was dependent on active TFIID. In contrast to other TATA-less promoters, purified TATA binding protein substituted for the TFIID complex and restored promoter activity to TFIID-inactivated nuclear extracts.

Identification of a tripartite basal promoter which regulates human terminal deoxynucleotidyl transferase gene expression.

In order to locate the promoter region of the human terminal deoxynucleotidyl transferase gene, serially truncated segments of the 5'-flanking region of the gene were cloned into a chloramphenicol acetyltransferase reporter vector. Transient transfection analyses of the terminal transferase-reporter gene constructs identified the basal promoter region within -34 to +40 base pairs relative to the transcription start site. Three promoter elements were defined in this region. The primary element is within 34 base pairs upstream of the transcription start site. The CAP site is 62 base pairs upstream of the translation start site. The secondary element involves sequences around the transcription start site. The third is located 25 base pairs downstream from the initiation site (+25 to +40). This tripartite basal promoter was not tissue specific; similar patterns of promoter activity were observed in terminal transferase expressing and non-expressing cells. Transfection analyses also indicated the presence of negative regulatory elements upstream of the basal promoter region, and these elements were preferentially active in cells expressing terminal transferase.

Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.

Human terminal deoxynucleotidyl transferase (TdT) was overexpressed in a baculovirus system. The pure recombinant enzyme was identical in size, activity, kinetic constants, and metal effects to native enzyme. Three amino acids, within either the putative nucleotide binding domain and part of a DNA polymerase consensus sequence, YGDTDSLF, or a TdT consensus sequence, GGFRRGK, were altered by site-directed mutagenesis. The four mutant forms of terminal transferase were also overexpressed in the baculovirus expression system and purified from Trichoplusia ni larvae by a monoclonal antibody affinity column and compared with wild-type enzyme with respect to thermostabilities, secondary structure, metal effects, and kinetic parameters. Three of the four mutants retained 3-16% of wild-type activity under varying metal conditions, and one of the mutants, D343E, consistently exhibited less than 0.2% of wild-type TdT activity with dATP and no activity with dGTP. All mutants had alterations in the Km for dATP. Variations in Km for dGTP were not as consistent. The Km for the other substrate, DNA initiator (dA)50) in the presence of dATP remained essentially the same as that of wild-type TdT for all mutants except D343E. The enzyme activity of all mutants was stimulated by Zn2+ at low concentrations, and this effect was diminished and reversed at higher concentrations of ZnSO4. All mutants still retained significant amounts of the secondary structure as measured by circular dichroism. These results indicated that the aspartic acid residue at position 343 is located at or near the active site and is critical for the nucleotide binding and catalytic activity.

Mutational analysis of active site residues of human adenosine deaminase.

Adenosine deaminase was overexpressed in a baculovirus system. The pure recombinant and native enzymes were identical in size, Zn2+ content, and activity. Five amino acids, in proximity to the active site, were replaced by mutagenesis. The altered enzymes were purified to homogeneity and compared to wild-type adenosine deaminase with respect to zinc content, enzymatic activity, and kinetic parameters. All but one of the alterations produced significant activity perturbations. Replacement of Cys262 produced a protein that retained at least 30-40% of wild-type activity. In contrast, replacements of His17, His214, His238, and Glu217 resulted in dramatic losses of enzyme activity. None of these mutants exhibited large variations in Km. The proteins produced from alterations of amino acids implicated in metal coordination were slightly activated by inclusion of Zn2+ throughout purification. These experiments confirm that in the active enzyme Zn2+ plays a critical role in catalysis, that a histidine or glutamate residue plays a mechanistic role in the hydrolytic deamination step, and that cysteine is not involved in the catalytic mechanism of adenosine deaminase. These data support the roles for these amino acid residues suggested from the x-ray structure of murine adenosine deaminase (Wilson, D. K., Rudolf, F. B., and Quicho, F. A. (1991) Science 252, 1278-1284).

Lack of functional significance of Cys227 and Cys234 in terminal deoxynucleotidyltransferase.

Identification of the three functional regions (catalytic, nucleotide substrate-binding, DNA substrate-binding) of the monofunctional template independent DNA polymerase terminal deoxynucleotidyltransferase has not been completely established. The potential participation of 2 amino acid residues, Cys227 and Cys234, has been controversial, and conflicting data have been published. To investigate the role of Cys227, the human terminal transferase cDNA was modified by site-directed mutagenesis to introduce a glycine codon at this position. Mutant and control wild-type human terminal transferase cDNAs had to be inserted into baculovirus genomes by homologous recombination and overexpressed in Trichoplusia ni insect larvae because terminal transferase cDNAs have not been successfully expressed in bacterial systems. The Cys227----Gly mutant and wild-type enzymes displayed similar km values for both the nucleotide (dGTP) and DNA initiator (dA50) substrates. The kcat for the mutant enzyme (0.56 s-1) was comparable to that of the native enzyme (0.58 s-1). Additionally, catalysis by both mutant and wild-type enzymes was stimulated by Zn2+. These results together with the observation that the amino acid residue at position 234 is not conserved across species indicated that neither Cys234 nor Cys227 is an essential residue in the active site of terminal transferase.

Regulation of terminal deoxynucleotidyl transferase gene expression in mice and men.

A nontemplate directed DNA polymerase, terminal deoxynucleotidyl transferase (terminal transferase) is expressed in a tissue-specific and development stage-specific manner. Its enzymatic properties and tissue localization have implicated the protein in development of normal immune function. Significant progress has been made in understanding the enzymology and important domains of this protein. More recently, studies have focused on regulation of the gene that codes for the protein in mice and humans. The murine gene has yielded to these studies more readily than the human gene. A murine basal promoter element has been identified along with several trans-acting protein factors that may regulate gene expression. In the human gene there is no evidence for a basal promoter element. Rather, the promoter exhibits tissue specific properties. The present article reviews recent developments in this field.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp221-Lys231 (peptide B8) and Cys234-Lys249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an alpha-helical array of 39 A which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase beta that has been implicated in the binding of DNA template.

Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellular adenosine deaminase mRNA, protein, and enzymatic activity were detected. The recombinant human adenosine deaminase represented 10% of the total cellular protein and exhibited a specific activity of 70 units/mg of protein in crude homogenate. This specific activity is 70-350 times greater than that exhibited by the enzyme in homogenates of the two most abundant natural sources of human adenosine deaminase, thymus and leukemic cells. When the recombinant virus was injected into insect larvae, the maximum recombinant enzyme was produced 4 days postinfection and represented about 2% of the total insect protein with a specific activity of 10-25 units/mg of protein. The recombinant human adenosine deaminase was purified to homogeneity from both insect cells and larvae and demonstrated to be identical to native adenosine deaminase purified from human cells with respect to molecular weight, interaction with polyclonal anti-adenosine deaminase antibody, and enzymatic properties. A pilot purification yielded 8-9 mg of homogeneous enzyme from 22 larvae. The production of large quantities of recombinant human adenosine deaminase in insect larvae is inexpensive and rapid and eliminates the need for specialized facilities for tissue culture. This method should be applicable to large-scale production of many recombinant proteins.

Tissue-specific expression of human terminal deoxynucleotidyl transferase is regulated at the transcriptional level.

Transcriptional regulation of expression of the terminal deoxynucleotidyl transferase gene in normal thymus and in differentiation arrested cells was demonstrated by analyzing steady-state levels of TdT RNA as well as the relative transcription rate of the gene. Terminal transferase transcripts were detected only in those cells and tissues that contained antigen and enzyme activity. The relative rates of transcription correlated with levels of mRNA as well as with levels of the protein. These data suggest that expression of this gene in normal and leukemic cells is modulated at the level of transcription.

Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation.

Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain.

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 1. Active site directed interactions with 8-azido-2'-deoxyadenosine 5'-triphosphate.

A photoaffinity analogue of dATP, 8-azido-2'-deoxyadenosine 5'-triphosphate (8-azido-dATP), was used to probe the nucleotide binding site of the non-template-directed DNA polymerase terminal deoxynucleotidyl transferase (EC 2.7.7.31). The Mg2+ form of 8-azido-dATP was shown to be an efficient enzyme substrate with a Km of 53 microM. Loss of enzyme activity occurred during UV photolysis only in the presence of 8-azido-dATP. At saturation (120 microM 8-azido-dATP), 54% of the protein molecules were modified as determined by inhibition of enzyme activity. Kinetic analysis of enzyme inhibition induced by photoincorporation of 8-azido-dATP indicated an apparent Kd of approximately 38 microM. Addition of 2 mM dATP to 120 microM 8-azido-dATP resulted in greater than 90% protection from photoinduced loss of enzyme activity. In contrast, no protection was observed with the addition of 2 mM dAMP. Enzyme inactivation was directly correlated with incorporation of radiolabeled 8-azido-dATP into the protein and UV-induced destruction of the azido group. Photoincorporation of 8-azido-dATP into terminal transferase was reduced by all purine and pyrimidine deoxynucleoside triphosphates of which dGTP was the most effective. The alpha and beta polypeptides of calf terminal transferase were specifically photolabeled by [gamma-32P]-8-azido-dATP, and both polypeptides were equally protected by all four deoxynucleoside triphosphates. This suggests that the nucleotide binding domain involves components from both polypeptides.

Initiator role of double stranded DNA in terminal transferase catalyzed polymerization reactions.

Binding of the 58 kDa monomer and 44 kDa alpha beta dimer forms of terminal deoxynucleotidyl transferase to double stranded DNA was demonstrated by gel retardation and tryptophan fluorescence quenching. The dissociation constants and cooperativity parameters were similar to those that have been determined for binding of these two forms of terminal transferase to single stranded DNA. However, the double stranded DNA binding site size of 10 nucleotides was half the size expected. The efficacy of blunt ended DNA as an initiator in the polymerization reaction catalyzed by terminal transferase was demonstrated by radiometric assays and product analyses on agarose gels. The initial reaction kinetics indicated that dGTP but not dATP was added efficiently to a blunt double stranded DNA 3' end. These results are correlated with current models for in vivo terminal transferase function.

Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region.

Human terminal deoxyribonucleotidyltransferase (nucleoside-triphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. "TATA" and "CAAT" sequences, respectively, were found to start at nucleotides -185 and -204, -328, and 465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements.

Interaction of terminal transferase with single-stranded DNA.

A 58-kDa monomer of terminal transferase was isolated from calf thymus using a monoclonal antibody affinity column. The enzymatic activity was comparable to that of the 44-kDa alpha beta dimer isolated by conventional methods. Binding of the two enzyme forms to single-stranded DNA was monitored by fluorescence. The site size of both forms was approximately 11 +/- 2 nucleotides. Binding of the 44-kDa alpha beta dimer to polydeoxyadenosine was examined under several conditions. The cooperativity parameter increased from about 90 in the presence of Mg2+ to 300-400 in the absence of Mg2+. The observed dissociation constant of 3-5 microM was essentially independent of salt concentration, whereas the intrinsic dissociation constant decreased about 5-fold in the presence of Mg2+. The binding parameters of the 58-kDa monomer were independent of buffer composition and were similar to those of the 44-kDa alpha beta dimer in the presence of Mg2+. These results indicate that the additional 14-kDa peptide sequences present in the high molecular mass monomer form are not part of the DNA-binding site of terminal transferase.

Immunoaffinity purification and fluorescence studies of human adenosine deaminase.

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.