Reevaluating limits of detection of 12 lateral flow immunoassays for the detection of Yersinia pestis, Francisella tularensis, and Bacillus anthracis spores using viable risk group-3 strains.

AIM: Rapid detection of biological agents in biodefense is critical for operational, tactical and strategic levels as well as for medical countermeasures. Yersinia pestis, Francisella tularensis, and Bacillus anthracis are high priority agents of biological warfare or bioterrorism and many response forces use lateral flow assays (LFAs) for their detection. Several companies produce these assays, which offer results in short time and are easy to use. Despite their importance, only few publications on the limits of detection (LOD) for LFAs are available. Most of these studies used inactivated bacteria or risk group-2 strains. As the inactivation process in previous studies might have affected the tests' performances, it was our aim in this study to determine and compare the LOD of several commercially available LFAs using viable risk group-3 strains. METHODS AND RESULTS: Lateral flow assays from four different companies for the detection of following bacteria were evaluated: Y. pestis, F. tularensis and B. anthracis spores. Two independent quantification methods for each target organism were applied, in order to ensure high quantification accuracy. LODs varied greatly between tests and organisms and ranged between 104 for Y. pestis-tests and as high as >109 for one B. anthracis-test. CONCLUSION: This work precisely determined the LODs of LFAs from four commercial suppliers. The herein determined LODs differed from results of previous studies. This illustrates the need for using accurately quantified viable risk group 3-strains for determining such LODs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work bridges an important knowledge gap with regard to LFA LOD. The LODs determined in this study will facilitate better assessment of LFA-results. They illustrate that a negative LFA result is not suited to exclude the presence of the respective agent in the analyzed sample.

Development and first use of a novel cylindrical ball bearing phantom for 9-DOF geometric calibrations of flat panel imaging devices used in image-guided ion beam therapy.

Image guidance during highly conformal radiotherapy requires accurate geometric calibration of the moving components of the imager. Due to limited manufacturing accuracy and gravity-induced flex, an x-ray imager's deviation from the nominal geometrical definition has to be corrected for. For this purpose a ball bearing phantom applicable for nine degrees of freedom (9-DOF) calibration of a novel cone-beam computed tomography (CBCT) scanner was designed and validated. In order to ensure accurate automated marker detection, as many uniformly distributed markers as possible should be used with a minimum projected inter-marker distance of 10 mm. Three different marker distributions on the phantom cylinder surface were simulated. First, a fixed number of markers are selected and their coordinates are randomly generated. Second, the quasi-random method is represented by setting a constraint on the marker distances in the projections. The third approach generates the ball coordinates helically based on the Golden ratio, ϕ. Projection images of the phantom incorporating the CBCT scanner's geometry were simulated and analysed with respect to uniform distribution and intra-marker distance. Based on the evaluations a phantom prototype was manufactured and validated by a series of flexmap calibration measurements and analyses. The simulation with randomly distributed markers as well as the quasi-random approach showed an insufficient uniformity of the distribution over the detector area. The best compromise between uniform distribution and a high packing fraction of balls is provided by the Golden section approach. A prototype was manufactured accordingly. The phantom was validated for 9-DOF geometric calibrations of the CBCT scanner with independently moveable source and detector arms. A novel flexmap calibration phantom intended for 9-DOF was developed. The ball bearing distribution based on the Golden section was found to be highly advantageous. The phantom showed satisfying results for calibrations of the CBCT scanner and provides the basis for further flexmap correction and reconstruction developments.

Hormonal control of GTP cyclohydrolase I gene expression and enzyme activity during color pattern development in wings of Precis coenia.

Color patterns of butterfly wings are composed of single color points represented by each scale. In the case of Precis coenia, at the end of pupal development, different types of pigments are synthesized sequentially in the differently colored scales beginning with white (pterins) followed by red (ommatins) and then black (melanin). In order to explain how formation of these different colors is regulated, we examined the expression of an mRNA-encoding guanosine triphosphate-cyclohydrolase I (GTP-CH I; EC 3.5.4.16), the first key enzyme in the biosynthesis of pteridines, during pigment formation in the wings of P. coenia. The strongest positive signal was recognized around pigment formation one day before butterfly emergence. This GTP-CH I gene expression is paralleled by GTP-CH I enzyme activity measured in wing extracts. We also investigated the effect of 20-hydroxyecdysone on the expression of GTP-CH I mRNA and the enzyme activity during color formation. The results strongly suggest that the onset and duration of the expression of a GTP-CH I mRNA is triggered by a declining ecdysteroid hormone titer during late pupal development.

Cloning and developmental expression of zebrafish GTP cyclohydrolase I.

GTP cyclohydrolase I (GCH) catalyses the conversion of GTP to dihydroneopterin triphosphate, initiating the pteridine pathway. The final product tetrahydrobiopterin (H4biopterin) is the cofactor for neurotransmitter synthesis and for tyrosine supply during melanogenesis. Sepiapterin accumulates as a pigment. We cloned the zebrafish gch cDNA, which encodes a protein highly homologous to other vertebrate sequences and characterised the recombinant enzyme. By in situ hybridisation, we found that gch is expressed in both the melanophore and xanthophore lineages during early development. gch-expression almost disappears after 3 days post-fertilisation (dpf), despite further sepiapterin synthesis. gch-transcripts are also located in catecholaminergic neurons, within the central nervous system and in the arch-associated neurons.

The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells.

Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 x anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcgamma-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUII-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUII to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcgamma-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis.

Development of the pteridine pathway in the zebrafish, Danio rerio.

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.

Simultaneous quantification of released succinic acid and a weakly basic drug compound in dissolution media.

A HPLC-method was developed to determine both fenoldopam, a weakly basic drug and succinic acid, a pH-adjuster for this drug in dissolution media. The usual assays for succinic acid were not applicable due to its low UV-absorption, the low pH-value of samples or the presence of buffer salts and fenoldopam. The described method is a simple non-ion-pair reversed phase HPLC-method using a fast scanning UV-detector and a PC software program for the quantification of both components. Succinic acid is detected at 205 nm and fenoldopam at 225 nm. The UV-spectrum is used to determine peak purity and to identify peaks (carried out at a 99.9% match). This is especially important as in some of the investigated samples an unknown peak elutes immediately after succinic acid, resulting in spurious high contents, if mistaken for succinic acid. The simple method accomplished the simultaneous quantification of both, succinic acid and fenoldopam, by an accurate, precise, specific and reproducible assay, with a linear range covering all concentrations relevant for dissolution testing. The method is stability indicating and can also be used for the quantification of fumaric acid, another pH-adjuster in dissolution media together with fenoldopam.

Phosphorylation of GTP cyclohydrolase I and modulation of its activity in rodent mast cells. GTP cyclohydrolase I hyperphosphorylation is coupled to high affinity IgE receptor signaling and involves protein kinase C.

GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hültner, L. , Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rödl, W. (1993) J. Biol. Chem. 268, 12544-12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivo labeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.

The pH-independent release of fenoldopam from pellets with insoluble film coats.

Various ratios of succinic acid to fenoldopam mesylate, ranging from 0:1 to 18:1 were incorporated in pellets and coated with 1.5-12% w/w Surelease. Even though the coating level did influence the rate and amount of fenoldopam release, the influence of the succinic acid to drug ratio was much more important and evident at all coating levels. Being a weakly basic drug, fenoldopam release ceased when testing in SIF for succinic acid to drug ratios of 0:1-4:1, with the end of release being more abrupt for the 0:1 than for the 4:1 ratio. Only for a succinic acid to drug ratio of > or =5 was fenoldopam release constant for 6-8 h and independent of the pH-value of dissolution media. For a thin coat of about 2.5% w/w Surelease, those pellets showed an ideal controlled release behaviour with release rates of about 5-10%/h and a total release of almost 80% in 8 h. The dissolution profiles of Surelease coated pellets with high succinic acid to drug ratios (> or =5) and different coating levels, were evaluated for best fits to commonly used kinetic models. Sustained release mechanisms are discussed according to best fit models. The quantification of the pH-adjuster succinic acid, released from pellets with an acid to drug ratio of < or =1 showed, that despite their failure as a controlled release system for fenoldopam, the investigated coats could control the release of succinic acid effectively at optimized coating levels. For increasing succinic acid to drug ratios (< or =4) succinic acid was released at an ever more constant rate and release rates, though still faster than the release rates of fenoldopam, decreased steadily for increasing ratios. At a 5:1 ratio finally release rates of succinic acid and fenoldopam were almost identical. Therefore those pellet cores were almost completely emptied during dissolution testing, with both fenoldopam and succinic acid leaving at a constant rate and a total release of about 80% each for a 2.5% Surelease coat, while lower succinic acid to drug ratios had failed to show any sustained release for such thin Surelease coats. A similar formulation with fumaric acid instead of succinic acid failed to show the desired release pattern, indicating that it is the presence of a sufficiently high amount of succinic acid rather than the presence of an acidic compound in general, that ensures fenoldopam solubility at higher pH-values.

Investigations on the influence of the type of extruder for pelletization by extrusion-spheronization. I. Extrusion behavior of formulations.

Three different extruders (the Alexanderwerk gravity feed roll extruder, the Gabler axial, single-screw extruder, and the NICA radial-screw extruder) were compared for their suitability for different placebo formulations and for fenoldopam pellets. A fourth extruder, the experimental ram extruder, was also included in some of the comparisons. Evaluation of the extrusion behavior of the three extruders showed differences as well as similarities among them, depending on the composition of the formulation. Although the NICA and Alexanderwerk units extruded all formulations successfully, the Gabler extruder failed to do so at a content of > 60% of soluble ingredients, such as lactose or mannitol. The extrudate surface improved for all extruders with an increase in water content of formulations, but was generally smoother for the Gabler than for the NICA or the Alexanderwerk units. A formulation with colloidal Avicel as spheronization aid showed an identical extrusion behavior for all of the investigated extruders. Of the three extruders, the Gabler unit showed the highest heat generation during extrusion, especially when extruding formulations with a low water content or high contents of soluble excipients. However, when the loss of water during extrusion or spheronization for various formulations was compared, only a two-way ANOVA test on the differences between the water content after extrusion and after spheronization showed a statistically significant difference between the Alexanderwerk or NICA and the Gabler extruder. The two-way ANOVA also proved that this difference is significant only for some formulations, e.g., lactose + Avicel PH 101 formulations, but not for Avicel PH 101 formulations.

Investigations on the influence of the type of extruder for pelletization by extrusion-spheronization. II. Sphere characteristics.

Three different extruders, the Alexanderwerk gravity-feed roll extruder, the Gabler axial, single-screw extruder, and the NICA radial-screw extruder, were compared for their suitability for different placebo formulations and for fenoldopam pellets. A fourth extruder, the experimental ram extruder, was also included in some of the comparisons. The successful spheronization of extrudates from each of these extruders requires the correct water content. This water content, however, is different for each of the formulations and for each extruder. Generally, the Gabler unit required the highest amounts of water for a successful spheronization, yielding > or = 90% between 710 and 1250 microns. The NICA unit needed much less water for the same formulation, and the Alexanderwerk unit required even less water than the NICA unit. Pellet sphericity was also strongly dependent on the correct water content of formulations, but was generally better for pellets produced with the Alexanderwerk or NICA units. A two-way ANOVA test for the individual formulations showed a significant difference in the mean particle size of batches produced with the NICA or the Alexanderwerk and the Gabler extruder. No significant differences could be found between any of the Alexanderwerk or NICA batches. Both extruders showed a linear dependence of the mean particle size on the water content of formulations, but the Gabler extruder showed an almost unchanged particle size over a wide range of water contents, provided that the formulation could be extruded successfully. Batches that were extruded on the NICA unit showed a significantly lower bulk density than comparable Alexanderwerk or Gabler batches. Comparing the true density of pellets, we found that significant differences could only be stated for Avicel PH 101 + water batches and only for the NICA/Gabler interaction. True density increased for all three extruders with increasing amounts of soluble components and with increasing water content. The NICA batches also exhibited a significant difference of the Hausner factor from the other two extruders, but no differences could be found in the friability of pellets.

Coordinate induction of tetrahydrobiopterin synthesis and nitric oxide synthase activity in chicken macrophages: upregulation of GTP-cyclohydrolase I activity.

Biosynthesis of nitric oxide (NO) and tetrahydrobiopterin (BH4) was investigated during cytokine-mediated activation of chicken macrophages. Monocyte derived macrophages and HD11 cells, a chicken macrophage cell line, constitutively synthesize BH4. Treatment of these cells with chicken macrophage activation factor (ChMAF) causes up to 10-fold increases of intracellular BH4 and of nitrite concentrations in the cell culture supernatant. Elevated BH4 levels correlate with an increase in GTP-cyclohydrolase I (GTP-CH) activity. Kinetic studies show a joint upregulation of GTP-CH activity and NO synthase activity first detectable 4 hr after stimulation. A corresponding increase in the mRNA for GTP-CH was detected by Northern blot analysis with a chicken GTP-CH specific cDNA probe. These results demonstrate that cytokine-induced BH4 synthesis by chicken macrophages is at least partially regulated through increased GTP-CH gene expression. The functional relevance of BH4 formation for NO production is shown by experiments using 2,4-diamino-6-hydroxypyrimidine (DAHP) as a specific inhibitor of GTP-CH. Monocyte derived macrophages stimulated in the presence of DAHP show a significant decrease in NO synthesis. The effect of DAHP was reversed by adding sepiapterin, which allows synthesis of BH4 through a salvage pathway.

[Treatment of unstable distal radius fractures with a small AO-fixateur externe not bridging the joint]. / Die Behandlung instabiler distaler Radiusfrakturen mit dem nicht gelenküberbrückenden kleinen AO-Fixateur externe.

This retrospective study investigated the stability of the small AO external fixator in a radioradial configuration used in the treatment of 42 mostly unstable distal radius fractures (types A-3.2, C-1.2 and C-2.1 according to the AO classification). No bone grafting was performed. All cases were documented with antero-posterior and lateral X-rays preoperatively, 0, 2 and 6 weeks postoperatively and after consolidation. Early functional aftertreatment was started one week after surgery. Thirty-six fractures showed a dorsal comminution, combined with osteopenia in 18 cases. The mean preoperative radial angle of 17 degrees was normalized to 25 degrees postoperatively. The mean preoperative volar angle of minus 30 degrees was reduced to 12 degrees postoperatively. None of these mean angles changed until consolidation. The external fixator was removed on average after 7 weeks (range 6-10 weeks) depending on the radiological fracture healing. The small AO external fixator in a radioradial configuration proved to be stable enough for early functional aftertreatment of the wrist. Its stability is related to a proper operative technique and correct indication (unstable Colles type with one or two distal fragments). Bone grafting is unnecessary if the external fixator is left in situ until bony consolidation.

Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum.

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM.

Cloning, sequencing and functional studies of the gene encoding human GTP cyclohydrolase I.

We have identified a genomic clone containing the 5' regulatory region of the gene GTP-CH encoding human GTP cyclohydrolase I. The transcription start point (tsp) was mapped by 5'-rapid amplification of cDNA ends (5'-RACE). The 2.6-kb region upstream from the tsp showed promoter activity when ligated upstream from a reporter gene. The truncation of approximately 2 kb of the promoter did not change expression activity, while a further removal of 243 bp halved the activity. The promoter contains CCAAT and TATA boxes. The GC-rich region close to the tsp, which contains several putative Sp1-responsive elements, is required for maximum promoter activity. Interferon-gamma treatment of B-cells transfected with reporter constructs had no influence on the expression activity.

Tetrahydropterins interfere with the G protein pathway in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum produces tetrahydrodictyopterin, the D-threo-isomer of tetrahydrobiopterin. During cyclic AMP coordinated aggregation, the G protein linked signalling pathway is involved in the regulation of the initial enzyme GTP cyclohydrolase I [M. Gütlich et al., Biochem. J. 314, 95-101 (1996)]. We now find that cyclic AMP stimulated binding of GTPgammaS to the membrane fraction is inhibited by tetrahydrodictyopterin and tetrahydrobiopterin. Inhibition was a function of GTPgammaS concentration and the analysis of the kinetic data pointed to a competitive type of inhibition. The inhibition of G protein activation was accompanied by a loss of adenylyl cyclase activation. Because the GTPgammaS- and G protein-dependent reduction of receptor affinity for cyclic AMP was also attenuated, we conclude that tetrahydrodictyopterin and tetrahydrobiopterin interfere with activation of G proteins by inhibiting GDP-GTP exchange.

Control of 6-(D-threo-1',2'-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity.

6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.

Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reverse-transcription polymerase chain reaction using a general set of degenerate primers.

GTP-cyclohydrolase I is the primary enzyme of tetrahydrobiopterin and folic acid biosynthesis. cDNA fragments of GTP-cyclohydrolase I were obtained from rainbow trout, chicken, the fungi Neurospora crassa, Phycomyces blakesleeanus and Saccharomyces cerevisiae, the cellular slime mold Dictyostelium discoideum, the phytoflagellate Euglena gracilis and the higher plant Mucuna hassjo using primers specific for conserved regions of the open reading frame and the reverse transcription polymerase chain reaction (RT-PCR) technique. A number of regions were found to be strictly conserved between unrelated eukaryotes. These regions may be essential for the function of GTP-cyclohydrolase I and are discussed with respect to the recently resolved crystal structure of the Escherichia coli enzyme.

Molecular characterization of HPH-1: a mouse mutant deficient in GTP cyclohydrolase I activity.

GTP cyclohydrolase I catalyzes the initial and rate limiting step of the biosynthesis of tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylation. The mouse mutant HPH-1, previously generated by chemical mutagenesis, shows a phenylketonuria due to decreased hepatic GTP cyclohydrolase I activity. We show that both parameters GTP cyclohydrolase I activity and tetrahydrobiopterin synthesis significantly increase after weaning, but remain reduced during the lifetime. In the wild type mouse (C57BL/6), interferon-gamma and kit ligand induce GTP cyclohydrolase I activity in primed T-cells and in bone marrow-derived mast cells, respectively. The same is true for the HPH-1 mutant, but the absolute values remain lower throughout. The open reading frame of GTP cyclohydrolase I is not affected by the hph-1 mutation as shown by sequencing. Northern blot analysis demonstrates a marked decrease in the steady state mRNA level specific for GTP cyclohydrolase I.

Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme.

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.