Accuracy of composite diagnostic standards for pneumococcal pneumonia in vaccine trials.

Because of a lack of gold standard diagnostics, a combination of multiple diagnostic tests, or composite diagnostic standard, has been used to measure pneumococcal pneumonia (PP) in pneumococcal vaccine trials. We estimated the accuracy of composite diagnostic standards for PP used in previous randomised controlled trials by simple formulas. A systematic literature review identified five eligible trials and all trials had used different combinations of diagnostic tests for PP. The estimated values of sensitivity and minimum specificity of composite diagnostic standards varied substantially between trials: 48.4% to 98.1% and 71.0% to 97.3%, respectively. Without standardizing the outcome measurements, pneumococcal vaccine efficacy estimates against PP are not comparable between trials and their pooled estimates are biased.

High incidence of acute traumatic spinal cord injury in a rural population in Japan in 2011 and 2012: an epidemiological study.

STUDY DESIGN: Retrospective questionnaire-based epidemiological study. BACKGROUND: Physicians treating acute traumatic spinal cord injury (SCI) in Japan noticed an increased occurrence of cervical SCI without skeletal injury. OBJECTIVE: To elucidate the precise epidemiology of acute cervical SCI with the aim of planning a prevention program. METHODS: Questionnaires were posted to all hospitals in Tokushima prefecture (around 780,000 inhabitants) to investigate the annual incidence of SCI in 2011 and 2012. RESULTS: The response rate was 79% in 2011 and 64% in 2012. The returned questionnaires reported on 95 patients in 2011 and 91 patients in 2012, with a mean age of 67.6 and 64.3 years and an annual incidence (per million population) of 121.4 and 117.1, respectively. More than two-thirds of the cases suffered cervical SCI without skeletal injury, and 61% of these were categorized as Frankel D neurological deficits due to low-energy impact as the main cause. CONCLUSION: The incidence of incomplete cervical SCI does appear to be increasing, and significant regional differences in the incidence of cervical SCI exist across Japan. We speculate that factors other than age are contributing to this increase.

www.elearnSCI.org: a global educational initiative of ISCoS.

OBJECTIVE: To develop a web-based educational resource for health professionals responsible for the management of spinal cord injury (SCI). The resource:www.elearnSCI.org is comprised of seven learning modules, each subdivided into various submodules. Six of the seven modules address the educational needs of all disciplines involved in comprehensive SCI management. The seventh module addresses prevention of SCI. Each submodule includes an overview, activities, self-assessment questions and references. DEVELOPMENT OF THE RESOURCE: Three hundred and thirty-two experts from The International Spinal Cord Society (ISCoS) and various affiliated societies from 36 countries were involved in developing the resource through 28 subcommittees. The content of each submodule was reviewed and approved by the Education and Scientific Committees of ISCoS and finally by an Editorial Committee of 23 experts. KEY FEATURES: The content of the learning modules is relevant to students and to new as well as experienced SCI healthcare professionals. The content is applicable globally, has received consumer input and is available at no cost. The material is presented on a website underpinned by a sophisticated content-management system, which allows easy maintenance and ready update of all the content. The resource conforms to key principles of e-learning, including appropriateness of curriculum, engagement of learners, innovative approaches, effective learning, ease of use, inclusion, assessment, coherence, consistency, transparency, cost effectiveness and feedback. CONCLUSION: www.elearnSCI.org provides a cost effective way of training healthcare professionals that goes beyond the textbook and traditional face-to-face teaching.

The importance of cellular environment to function of the CD44 matrix receptor.

Much has been learned recently by experimental manipulation of the structure of CD44 and assessment of the resulting functions. However, even greater structural variation is naturally introduced by CD44-bearing cells. A structural model is now available for the portion of CD44 that recognizes hyaluronan, but it is clear that all domains of the molecule influence CD44 functions.

Glycosylation of CD44 negatively regulates its recognition of hyaluronan.

Although CD44 is expressed on a wide variety of cell types, few of them use it to recognize the ligand hyaluronan (HA). A glycosylation-defective clone of Chinese hamster ovary cells (Lec 8) bound HA, demonstrating that complete processing of glycoproteins with addition of a full complement of sialic acid is not required. On the contrary, subsequent findings revealed that complex sugars on CD44 can actually inhibit ligand recognition. Two subclones of wild-type Chinese hamster ovary cells with similar amounts of surface CD44 were isolated on the basis of HA binding and found to differ with respect to CD44 size as well as staining with fluorescent lectins. Treatment of the nonbinding clone with tunicamycin reduced the size of the protein and allowed the cells to recognize HA via CD44. This function was also induced by treatment with deglycosylating enzymes (either a mixture of endoglycosidase F and N-glycosidase F or neuraminidase alone). A possible role for glycosylation in regulation of adhesion was then sought with a series of normal and transformed murine cells. Disruption of glycosylation or treatment with deglycosylating enzymes did not induce ligand binding in an interleukin 7-dependent pre-B cell line, and splenic B cells also appeared to be in an inactive state. Some normal B cells acquired the ability to recognize HA after stimulation with lipopolysaccharide or interleukin 5 and had distinctive surface characteristics (loss of immunoglobulin D and acquisition of CD43). An additional subset of activated cells might have been in a transitional state, because the cells bound ligand after neuraminidase treatment. The ligand-binding ability of a purified CD44-immunoglobulin fusion protein dramatically increased after neuraminidase treatment. Thus, differential glycosylation of this molecule is sufficient to influence its recognition function. Cell adhesion involving HA can be regulated by multiple mechanisms, one of which involves variable glycosylation of CD44.

Transgenic mice expressing a B cell growth and differentiation factor gene (interleukin 5) develop eosinophilia and autoantibody production.

Interleukin 5 (IL-5) has been suggested to be involved in the growth and differentiation of B cells and eosinophils. Especially, Ly-1+ B cells, which have been considered to produce autoantibodies, are selectively developed by this lymphokine in long-term bone marrow culture. To envisage the possible engagement of IL-5 in the development of these cells in vivo, transgenic mice carrying the mouse IL-5 gene ligated with a metallothionein promoter were generated. Transgenic mice carrying the IL-5 gene exhibited elevated levels of IL-5 in the serum and an increase in the levels of serum IgM and IgA. A massive eosinophilia in peripheral blood, bone marrow, and spleen, and an infiltration of muscle and liver with eosinophils, were observed. When cadmium-containing saline was injected intraperitoneally into transgenic mice, IL-5 production was augmented about five times within 24 h, and a distinctive Ly-1+ B cell population became apparent in the spleen after 5 d. IL-5 receptors were detected on those cells by monoclonal antibodies against IL-5 receptors. Another interesting finding in these transgenic mice was an increase in polyreactive anti-DNA antibodies of IgM class. It is suggested, therefore, that aberrant expression of the IL-5 gene may induce accumulation of Ly-1+ B cells and eosinophils. Furthermore, this IL-5 transgenic mouse can be a model mouse for eosinophilia, and we can determine the role of IL-5 in the differentiation of Ly-1+ B cells and eosinophils by using this mouse.

A crucial role of sialidase Neu1 in hyaluronan receptor function of CD44 in T helper type 2-mediated airway inflammation of murine acute asthmatic model.

CD44 is a highly glycosylated cell adhesion molecule that is involved in lymphocyte infiltration of inflamed tissues. We have demonstrated previously that sialic acid residues of CD44 negatively regulates its receptor function and CD44 plays an important role in the accumulation of T helper type 2 (Th2) cells in the airway of a murine model of acute asthma. Here we evaluated the role of sialidase in the hyaluronic acid (HA) receptor function of CD44 expressed on CD4+ T cells, as well as in the development of a mite antigen-induced murine model of acute asthma. Splenic CD4+ T cell binding of HA was examined with flow cytometry. Expression of sialidases (Neu1, Neu2, Neu3 and Neu4) in spleen cells was evaluated by quantitative real-time reverse transcription-polymerase chain reaction. Airway inflammation and airway hyperresponsiveness (AHR) were evaluated in the asthmatic Neu1-deficient mouse strain SM/J model. Splenic CD4+ T cells from asthmatic model mice displayed increased HA receptor activity of CD44 after culture with the antigen, along with characteristic parallel induction of sialidase (Neu1) expression. This induction of HA binding was suppressed significantly by a sialidase inhibitor and was not observed in SM/J mice. Th2 cytokine concentration and absolute number of Th2 cells in the bronchoalveolar lavage fluid, and AHR were decreased in SM/J mice. In conclusion, HA receptor activity of CD44 and acute asthmatic reactions, including Th2-mediated airway inflammation and AHR, are dependent upon Neu1 enzymatic activity. Our observation suggests that Neu1 may be a target molecule for the treatment of asthma.

Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

Two electrogenic mechanisms contributing to the 560 nm absorption changes in intact Bryopsis chloroplasts.

Light -induced absorbance changes at 560 nm in dark-adapted intact chloroplasts of the green alga, Bryopsis maxima were studied in the time range of 200 ms. The initial rise of the 560 nm signals consists of two major components which are both electrochromic absorbance changes of the carotenoids, siponein and/or siphonaxanthin, but different in mechanisms of the field formation. The first component (component S) is related to electron transport since it was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and showed at light-intensity dependence similar to that of electron transport in chloroplasts. In the presence of DCMU, component S could be restored on addition of proton-transporting electron donors such as reduced 2.6-dichlorophenol indophenol and phenazine methosulfate, but not on addition of N,N,N',N'-tetramethyl-p-phenylenediamine which does not carry protons with electrons (Trebst, A. (1974) Annu. Rev. Plant Physiol. 25, 423--458). We propose that component S is due to the electric field set up by the proton translocation across the thylakoid membrane. The second component (component R) was resistant to DCMU and DBMIB. The light-intensity dependency of component R was similar to that of cytochrome f photooxidation which showed saturation at a relatively low light intensity. The magnitude of component R was markedly reduced by phenylmercuric acetate, suggesting the participation of ferredoxin and ferredoxin-NADP oxidoreductase in the mechanism of the field formation responsible for this component. In the presence of DCMU and phenylmercuric acetate, time courses of the 560 nm changes paralleled those of cytochrome f changes. These results indicate that component R is due to the electric field formed between oxidized cytochrome f and other intersystem electron carriers located in the inner part of the thylakoid membrane and reduced electron acceptors of Photosystem I situated on the membrane surface. The complex natures of the 560 nm changes, as well as the contributions of Photosystems I and II to the absorbance changes, are explained in terms of the two electrogenic mechanisms.

Light-induced changes of C-550 and fluorescence yield in ultraviolet-irradiated chloroplasts at room temperature.

Effects of ultraviolet irradiation of chloroplasts on several photochemical reactions mediated by Photosystem II were studied at room temperature. The Hill activity and fluorescence of variable yield were decreased by ultraviolet irradiation in parallel. The activity of 2,6-dichlorophenolindophenol (DCIP) photoreduction in irradiated chloroplasts was only slightly recovered by addition of diphenylcarbazide, an electron donor for Photosystem II. No restoration of the original fluorescence yield was observed on addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or preincubating the irradiated chloroplasts with dithionite. Photobleaching of C-550 was much more resistant than the Hill activity or fluorescence of variable yield toward ultraviolet irradiation. Chloroplasts, of which the Hill activity and variable fluorescence had mostly been eliminated by ultraviolet irradiation, still showed C-550 photobleaching, which was in magnitude more than 50% of that in the original unirradiated chloroplasts. C-550 was shown not to respond to membrane potential. The photobleaching of C-550 is sensitized by Photosystem II and proceeds with unaltered quantum efficiency in ultraviolet-irradiated chloroplasts. These observations are interpreted to indicate that C-550 is not identical with Q, the hypothetical quencher of fluorescence, and that C-550 is a better indicator for the primary photoact in Photosystem II than Q.

Purification and characterization of sepiapterin reductase from rat erythrocytes.

Sepiapterin reductase from rat erythrocyte hemolysate was purified 2000-fold to apparent homogeneity with 30% yield. The specific activity of the purified enzyme was 18 units/mg protein, and its molecular weight was 55 000. The enzyme consists of two identical subunits, each of which has a molecular weight of 27 500. The enzyme showed a single peak by isoelectric focusing with a pI of 4.9 and partial specific volume of 0.73 cm3/g. The amino acid composition was determined. pH optimum of the enzyme was 5.5. The equilibrium constant of 2.2.10(9) of the enzyme showed that the equilibrium lies much in favor of dihydrobiopterin formation from sepiapterin in rat erythrocytes. From steady-state kinetic measurements, ordered bi-bi mechanism was proposed to the reaction of sepiapterin reductase in which NADPH binds to free enzyme and sepiapterin binds next. NADP+ is released after the release of dihydrobiopterin. The Km values for sepiapterin and NADPH were 15.4 microM and 1.7 microM, respectively, and the Vmax value was 21.7 mumol/min per mg.

Carbonyl reductase activity of sepiapterin reductase from rat erythrocytes.

A homogeneous preparation of sepiapterin reductase, an enzyme involved in the biosynthesis of tetrahydrobiopterin, from rat erythrocytes was found to be responsible for the reduction with NADPH of various carbonyl compounds of non-pteridine derivatives including some vicinal dicarbonyl compounds which were reported in the previous paper (Katoh, S. and Sueoka, T. (1984) Biochem, Biophys. Res. Commun. 118, 859-866) in addition to the general substrate, sepiapterin (2-amino-4-hydroxy-6-lactoyl-7,8-dihydropteridine). The compounds sensitive as substrates of the enzyme were quinones, e.g., p-quinone and menadione; other vicinal dicarbonyls, e.g., methylglyoxal and phenylglyoxal; monoaldehydes, e.g., p-nitrobenzaldehyde; and monoketones, e.g., acetophenone, acetoin, propiophenone and benzylacetone. Rutin, dicoumarol, indomethacin, and ethacrynic acid inhibited the enzyme activity toward either a carbonyl compound of a non-pteridine derivative or sepiapterin as substrate. Sepiapterin reductase is quite similar to general aldo-keto reductases, especially to carbonyl reductase.

Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus. II. The psaE gene product has a role to promote interaction between the terminal electron acceptor and ferredoxin.

Function of a subunit polypeptide (the psaE gene product) of Photosystem I (PS I) reaction center complexes was investigated by comparing the reactivity of the reduced iron-sulfur centers (FA/FB)- with ferredoxin among Synechococcus PS I complexes which had been variously depleted of this polypeptide. Ferredoxin at or below 1 microM can accept electrons from (FA/FB)- effectively competing with the back reaction between P-700+ and (FA/FB)- in the thylakoid membranes and PS I complexes that contained all the eight small subunits. The high reactivity of (FA/FB)- with low concentrations of ferredoxin was observed in PS I complexes which contain only the products of psaC, psaD and psaE genes but not in complexes which carry the psaC, psaD, psaL and psaK gene products but no psaE gene product. Varied amounts of the psaE gene product were extracted by treatment with different concentrations of a cationic detergent, dodecyltrimethylammonium bromide, and 2.5 M NaCl. The solubilized polypeptide was then reconstituted to the depleted complexes. The magnitudes of the back reaction that could be suppressed by addition of ferredoxin at or below 1 microM were well correlated to the amounts of the psaE polypeptide remained bound or rebound to the complexes. It is concluded that the product of the psaE gene has a role to promote the interaction between the terminal bound electron acceptor and ferredoxin. A high autooxidizability of (FA/FB)- and contrasting effects of lipophilic cations and anions on the rate of the back reaction from (FA/FB)- to P-700+ were also reported.

The effect on photosynthetic electron transport of temperature-dependent changes in the fluidity of the thylakoid membrane in a thermophilic blue-green alga.

Various electron transport reactions in cell or isolated thylakoid membranes of the thermophilic blue-green alga, Synechococcus sp. were measured at different temperatures between 72 and 3 degrees C. They are classified into two groups with respect to their temperature dependency. The first group involves cytochrome 553 photooxidation, methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol as electron donor and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea-resistant ferricyanide photoreduction determined in the presence or absence of silicomolybdate. The Arrhenius plot of these reactions showed a single straight line with the activation energy of about 10 kcal/mol throughout wide temperature ranges studied. Methyl viologen photoreduction with water as electron donor, reduction of flash-oxidized cytochrome 553, ferricyanide photoreduction and photosynthetic O2 evolution form the second group. Their arrhenius plots are characterized by discontinuities or breaks at about 30 and 10 degrees C, which respectively correspond to the upper and lower boundaries of the lateral phase separation of the membrane lipids. The first group reactions represent short spans of electron transport which are mediated either by Photosystem I or Photosystem II alone and not related to plastoquinone, whereas all the reactions of the second group involve plastoquinone. It is concluded therefore that the membrane fluidity affect electron transport specifically at the region of plastoquinone. It is proposed that the reaction center chlorophyll-protein complexes of both Photosystems I and II are closely associated with related electron carrier proteins to form functional supramolecular assemblies so that electron transfer within such a cluster of proteins proceeds independently of the phase changes in the membrane lipids. On the other hand, the role of plastoquinone as a mobile electron carrier mediating electron transfer from the protein assembly of Photosystem II to that of Photosystem I through the fluid hydrophobic matrix of the membranes is highly sensitive to the physical state of the membrane lipids.

Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus. I. Is the psaF gene product required for oxidation of cytochrome c-553?

Photosystem I (PS I) reaction center complexes isolated from the thermophilic cyanobacterium Synechococcus elongatus with nonionic detergents, digitonin or sucrose monolaurate, contained eight small subunit polypeptides. Two of the small polypeptides were identified by analysis of their N-terminal amino-acid sequences as the psaF and psaE gene products. Treatment with a cationic detergent, cetyltrimethylammonium bromide, resulted in depletion of five small subunits including the psaF gene product. Five PS I complexes isolated with an anionic detergent, sodium dodecylsulfate, contained zero to four small subunits but were all depleted of the psaF polypeptide. The function of the psaF gene product was examined by measuring reduction kinetics of flash-oxidized P-700 in the presence of different concentrations of cytochrome c-553. Oxidized P-700 was rapidly reduced by the reduced cytochrome in all the PS I complexes that contained, at least, the psaC and psaD polypeptides and the second-order rate constants of electron transfer from cytochrome c-553 to P-700 were essentially the same between PS I complexes that contained the psaF polypeptide and those that lost this polypeptide. Thus, the psaF polypeptide is not required for the bimolecular reaction between P-700 and cytochrome c-553. Mg2+ had a moderate stimulating effect on the rate of P-700 reduction whether PS I complexes were associated with the psaF gene product or not. The function of this subunit polypeptide is discussed.

Two regions of the Mn-stabilizing protein from Synechococcus elongatus that are involved in binding to photosystem II complexes.

Limited proteolysis of the Mn-stabilizing protein (MSP) from the thermophilic cyanobacterium Synechococcus elongatus with chymotrypsin, trypsin or lysylendopeptidase that yielded four major polypeptides of 26 kDa, 22 kDa, 15 kDa and 11 kDa on denaturing gel electrophoresis resulted in total loss of the binding capacity of the protein to PSII complexes. Analyses of electrophoretic patterns and amino-terminal sequences of the proteolytic products revealed that the three proteases specifically cleaved the protein at a site between Phe156 and Gly163 or between Arg184 and Ser191. Site-directed mutagenesis was used to construct two mutant MSPs that had a nick between Phe156 and Leu157, a chymotrypsin-cleavage site, and Met before Leu157 or in place of Leu157. The two mutant proteins failed to bind to PSII complexes, although they largely retained ordered secondary structure and comigrated with the wild-type proteins in non-denaturing gel electrophoresis. The loss of the protein binding can be ascribed to introduction of a nick because a mutant protein that had Met in place of Leu157 but no nick was able to specifically bind to the functional site of PSII complexes and restore the oxygen-evolving activity as effectively as the wild-type protein. In contrast, a mutant MSP with Met inserted between Phe156 and Leu157 bound only weakly and non-specifically to PSII complexes and failed to reactivate oxygen evolution. Thus, the binding of the protein to the functional site of the PSII complex was highly sensitive to a small structural change that was caused by cleavage or insertion of a single amino acid residue between Phe156 and Leu157. The results suggest that the Phe156-Gly163 and Arg184-Ser191 sequences of the cyanobacterial MSP are regions for interaction with PSII complexes.

Dyspropterin, an intermediate formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin.

The structure of dyspropterin, a new name given to an intermediate which is formed from dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin, has been studied. Sepiapterin reductase (EC 1.1.1.153) was found to reduce dyspropterin to tetrahydrobiopterin in the presence of NADPH. Several lines of evidence showing the formation of tetrahydrobiopterin have been presented. Stoichiometric analysis revealed that there is a 1:2 relationship between the production of biopterin and the oxidation of NADPH during the reductase-catalyzed reduction of dyspropterin. The tetrahydrobiopterin production from dyspropterin was enhanced by dihydropteridine reductase (EC 1.6.99.7). Dyspropterin could also serve as a cofactor in phenylalanine hydroxylase (EC 1.14.16.1) system. These results are consistent with the view that dyspropterin is 6-(1,2-dioxopropyl)-5,6,7,8-tetrahydropterin. Based on our findings, the biosynthetic pathway of tetrahydrobiopterin from dihydroneopterin triphosphate has been discussed.

Nucleotide sequence of the Mn-stabilizing protein gene of the thermophilic cyanobacterium Synechococcus elongatus.

The nucleotide sequence of the psbO gene encoding the extrinsic 33 kDa protein (the Mn-stabilizing protein) from the thermophilic cyanobacterium Synechococcus elongatus was determined. The deduced amino acid sequence consisted of 272 residues; 26 for the signal peptide and 246 for the mature protein. The amino acid sequences of nine proteolytic peptides from the isolated protein completely agreed with the deduced amino acid sequence. Several unique variations of amino acids were found in the primary structure, of which some may be related to the high thermostability of the protein.

Secondary structure and thermostability of the photosystem II manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus.

The secondary structure of the manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus in solution was investigated by Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopies. Both methods showed a high proportion of disordered structure (40-43%) and a relatively small amount of beta-sheet (23-24%) and alpha-helix (17-19%). The conformation of the protein remained essentially unchanged at temperatures up to 70 degrees C. Unfolding of the protein occurred at higher temperatures and FT-IR spectroscopy revealed that beta-sheet was more strongly unfolded than alpha-helix at 76 degrees C. The protein largely lost the ordered secondary structures at 90 degrees C, but, when cooled down to 30 degrees C, regained its original conformation. Thus, the cyanobacterial protein is very thermostable and its denaturation at an extremely high temperature is reversible.

Dihydropteridine reductase and tetrahydropterin in Crithidia fasciculata cells.

Dihydropteridine reductase was found in extracts of Crithidia fasciculata and was demonstrated by the fact that the enzyme required both quinonoid-dihydropterin and NADH as substrates. 7,8-Dihydropterin and dihydrofolate failed to serve as substrates; tetrahydropterin was formed as the reaction product. The molecular weight of the enzyme was estimated to be about 55 000 by Sephadex G-100 gel filtration. NADH was more effective than NADPH as substrate for the enzyme. Tetrahydropterin (1.35 nmol tetrahydrobiopterin equivalents/g cells) was also detected in C. fasciculata.