Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures.

The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.

Education-corrected CERAD identifies MCI and dementia in Parkinson's disease.

OBJECTIVES: This study examined whether controlling for educational background in the CERAD cognitive screening battery would affect the likelihood of patients with Parkinson's disease to fulfill criteria for mild cognitive impairment (PD-MCI) and dementia (PDD). MATERIALS & METHODS: One-hundred seventeen patients with PD were studied. Cognitive impairment was determined as two subtest scores falling below either the standard cutoff scores or education-corrected cutoff scores. The presence of dementia was determined by clinical interview or Clinical Dementia Rating. Patients were then classified as PD-MCI and PDD according to cognitive test performance and presence/absence of dementia. RESULTS: The number of cognitively impaired patients (PD-MCI or PDD) was significantly higher when education-controlled cutoff scores were used (62.5% vs 38%). Correspondingly, the number of false negatives (demented PD patients performing normally in CERAD) was significantly lower when education-corrected cutoff scores were used (4% vs 10%). CONCLUSIONS: Controlling for education increases the sensitivity of the CERAD for PD-MCI and PDD.

Five-year follow-up of 11C-PIB uptake in Alzheimer's disease and MCI.

PURPOSE: The aim of this study was to evaluate the longitudinal changes in [(11)C]PIB uptake in mild cognitive impairment (MCI) and Alzheimer's disease (AD) over a long-term follow-up. METHODS: Six AD patients, ten MCI patients and eight healthy subjects underwent a [(11)C]PIB PET scan at baseline and at 2 and 5 years. The clinical status of the MCI patients was evaluated every 6 months. RESULTS: The MCI group showed a significant increase in [(11)C]PIB uptake over time (p < 0.001), with a similar increase from baseline to 2 years (4.7% per year) and from 2 to 5 years (5.0% per year). Eight MCI patients (80%) converted to AD, and two of these patients showed a normal [(11)C]PIB scan at baseline but increased uptake later. There was an increase in [(11)C]PIB uptake with time in the AD group (p = 0.02), but this did not significantly differ from the change in the control group. CONCLUSION: Our results revealed a significant increase in amyloid load even at the time of AD diagnosis in some of the MCI patients who converted. A positive [(11)C]PIB scan at baseline in MCI patients strongly predicted future conversion to AD but a negative PIB scan in MCI patients did not exclude future conversion. The results suggest that there is wide individual variation in the brain amyloid load in MCI, and in the course of amyloid accumulation in relation to the clinical diagnosis of AD.

Clofarabine in combination with a standard remission induction regimen (cytosine arabinoside and idarubicin) in patients with previously untreated intermediate and bad-risk acute myelogenous leukemia (AML) or high-risk myelodysplastic syndrome (HR-MDS): phase I results of an ongoing phase I/II study of the leukemia groups of EORTC and GIMEMA (EORTC GIMEMA 06061/AML-14A trial).

This study aims to determine the maximum tolerated dose (MTD) of clofarabine combined with the EORTC-GIMEMA 3 + 10 induction regimen (idarubicin + cytosine arabinoside) in adults with untreated acute myelogenous leukemia or high-risk myelodysplastic syndrome. In this phase I trial, 25 patients (median age 56 years) received 5 days of clofarabine as 1-h infusion (arm A) or push injection (arm B) at the dose level of 5 × 10 or 5 × 15 mg/m(2)/day in an algorithmic dose escalation 3 + 3 design. A consolidation course (intermediate dose cytosine arabinoside, idarubicin) was planned for patients in complete remission (CR). Primary endpoint was safety and tolerance as measured by dose limiting toxicity (DLT); secondary endpoints were response rate, other grade III/IV toxicities, and hematological recovery after induction and consolidation. Five DLTs were observed (in arm A: one DLT at 10 mg/m(2)/day, three at 15 mg/m(2)/day; in arm B: one DLT at 15 mg/m(2)/day). Three patients receiving 15 mg/m(2)/day were withdrawn due to adverse events not classified as DLT. Prolonged hypoplasia was observed in five patients. CR + complete remission with incomplete recovery were achieved in 21 patients (11/12 (92 %) receiving clofarabine 10 mg/m(2)/day; 10/13 (77 %) receiving clofarabine 15 mg/m(2)/day). Clofarabine, 5 × 10 mg/m(2)/day, resulted in one DLT and no early treatment withdrawals. MTD of clofarabine combined with cytosine arabinoside and idarubicin is 5 × 10 mg/m(2)/day.

Apathy is associated with activities of daily living ability in Parkinson's disease.

BACKGROUND: Neuropsychiatric symptoms and impairment in performing activities of daily living (ADL) in patients with Parkinson's disease (PD) are strong predictors of the overall caregiver burden and they increase the risk for nursing home admission of the patients. The purpose of the present study was to assess the association of neuropsychiatric symptoms and ADL functions in PD. METHODS: A total of 73 community-dwelling PD patients were studied. The mean age of the patient group was 65 years and the mean disease duration was 9 years. The Neuropsychiatric Inventory was used to measure neuropsychiatric symptoms, and ADL abilities were measured by the Alzheimer's Disease Cooperative Study-Activities of Daily Living Inventory. RESULTS: The prevalence of neuropsychiatric symptoms in patients with PD was 73%. The most common symptoms were depression, anxiety, irritability, apathy and agitation. ADL ability correlated significantly with apathy (p < 0.002) even when adjusted for motor symptoms. CONCLUSION: Apathy was significantly associated with ADL in PD. The result indicates that more attention should be paid to identifying apathy and targeting therapeutic interventions.

CERAD test performance and cognitive impairment in Parkinson's disease.

OBJECTIVES: Many patients with Parkinson's disease (PD) develop mild cognitive impairment (PD-MCI) and dementia (PDD). The Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neurocognitive test battery was originally developed to identify early Alzheimer's disease, but it has become a widely used screening instrument also for other types of dementia. The aim of the study was to examine differences in CERAD test performances between cognitively intact and impaired PD patients. MATERIALS AND METHODS: Eighty-eight PD patients participating in a rehabilitation course were studied. The Clinical Dementia Rating (CDR) was used to assess cognitive impairment. Sixty-six patients were cognitively intact and 22 had cognitive impairment (≥1 in two or more domains or a sum of boxes score of ≥3). The Finnish CERAD test battery was used to measure cognitive functions in seven different domains (language functions, verbal learning, visuospatial functions, delayed recall, memory consolidation, recognition memory, and executive functions). RESULTS: There were significant differences between the cognitively intact and impaired patients in six CERAD subtests (wordlist learning sum, wordlist delayed recall, constructional praxis recall, clock drawing, verbal fluency and constructional praxis copy) when controlling for covariates (disease duration, motor symptoms, age, and education). No differences were observed in memory consolidation scores. CONCLUSIONS: The results indicate that mild cognitive impairment in PD is related to deficits in memory, executive functions, and visuospatial functions. The memory deficit is non-amnestic and does not entail accelerated forgetting. CERAD shows promise in identifying PD patients with cognitive impairment and increased risk of dementia.

A systemic review of toxic death in clinical oncology trials: an Achilles' heel in safety reporting revisited.

BACKGROUND: Toxic death is defined as study treatment-related mortality and as such is considered as an iatrogenic death. This belongs to unnatural death where an autopsy is advised. Until now, conventional autopsy is the gold standard to discriminate between pre- and post-mortem discrepancies. METHODS: The consequences of lack of systematically performing an autopsy will be explored in the setting of oncological clinical trials. RESULTS: During more than one decade, 6428 Serious Adverse Events have been registered in the EORTC Safety database on a total of 34 734 subjects. The number of deaths were 764 (mortality rate of 2.2%) whereof 255 (rate of 0.7%) toxic deaths. In 89.8% of these toxic deaths, no autopsy has been done; in 25.1% (64 cases) an inconsistent cause of death was found based on studying of the medical narrative. The autopsy rate was only 10.2% (26 out of 255) and, in 46.2% of the performed autopsies, there was a clinical pathological discrepancy. CONCLUSION: When no autopsy is performed, there is a high risk for a wrong diagnosis in case of suspected toxic death. The high discrepancy rate, possibly due to a low autopsy rate, shows that toxic death is an Achilles' heel in iatrogenic mortality.

Efficacy and safety of bexarotene combined with psoralen-ultraviolet A (PUVA) compared with PUVA treatment alone in stage IB-IIA mycosis fungoides: final results from the EORTC Cutaneous Lymphoma Task Force phase III randomized clinical trial (NCT00056056).

BACKGROUND: Psoralen plus ultraviolet A (PUVA) is the standard treatment for early stages of mycosis fungoides. There have been no adequate randomized controlled trials with sufficient power comparing this modality with other therapies. OBJECTIVE: To assess disease response and to compare the response rates of patients treated with PUVA alone or PUVA and bexarotene. METHODS: EORTC 21011 (NCT 00056056) was a randomized phase III study comparing combined bexarotene (Targretin(®) ) and PUVA vs. PUVA alone in patients with stage IB and IIA mycosis fungoides (MF). The primary endpoint was the overall response rate [complete clinical response (CCR) plus partial response (PR)]. RESULTS: The study was prematurely closed due to low accrual after 93 of 145 required patients (65%) were randomized. Of the 93 randomized patients, 87 started treatment, 41 received PUVA and 46 received PUVA + bexarotene. Total UVA doses received were 107 J cm(-2) (range 1·4-489·9) in the PUVA arm vs. 101·7 J cm(-2) (0·2-529·9) in the combination arm. The safety profile was acceptable with few grade 3-4 toxicities observed in either arm. More drop-outs due to toxicity were observed in the combination arm compared with the PUVA-alone arm. The best overall response (CCR + PR) rate was 71% for PUVA alone and 77% for the combination arm (P = 0·57). The median duration of response was 9·7 months for PUVA vs. 5·8 months for the combination arm (P = 0·33). CCR was seen in 25 patients of whom 10 received PUVA alone (CCR 22%) and 15 received combination therapy (CCR 31%) (P = 0·45). CCR was sustained in 25% of patients regardless of therapy. There was a trend towards fewer PUVA sessions needed to achieve CCR in the combination arm (median 22) compared with the PUVA arm (median 27·5) (P = 0·11). Similarly, a trend towards lower UVA dose required to achieve CCR in the combination arm (median 55·8 J cm(-2) ) compared with the PUVA arm alone (median 117·5 J cm(-2) ) (P = 0·5) was observed. CONCLUSIONS: No significant difference in response rate or response duration was observed in this study. However, there was a trend towards fewer PUVA sessions and lower UVA dose required to achieve CCR in the combination arm (PUVA + bexarotene) but this did not achieve statistical significance due to insufficient power.

Deep amoA amplicon sequencing reveals community partitioning within ammonia-oxidizing bacteria in the environmentally dynamic estuary of the River Elbe.

The community composition of betaproteobacterial ammonia-oxidizing bacteria (ß-AOB) in the River Elbe Estuary was investigated by high throughput sequencing of ammonia monooxygenase subunit A gene (amoA) amplicons. In the course of the seasons surface sediment samples from seven sites along the longitudinal profile of the upper Estuary of the Elbe were investigated. We observed striking shifts of the ß-AOB community composition according to space and time. Members of the Nitrosomonas oligotropha-lineage and the genus Nitrosospira were found to be the dominant ß-AOB within the river transect, investigated. However, continuous shifts of balance between members of both lineages along the longitudinal profile were determined. A noticeable feature was a substantial increase of proportion of Nitrosospira-like sequences in autumn and of sequences affiliated with the Nitrosomonas marina-lineage at downstream sites in spring and summer. Slightly raised relative abundances of sequences affiliated with the Nitrosomonas europaea/Nitrosomonas mobilis-lineage and the Nitrosomonas communis-lineage were found at sampling sites located in the port of Hamburg. Comparisons between environmental parameters and AOB-lineage (ecotype) composition revealed promising clues that processes happening in the fluvial to marine transition zone of the Elbe estuary are reflected by shifts in the relative proportion of ammonia monooxygenase sequence abundance, and hence, we propose ß-AOB as appropriate indicators for environmental dynamics and the ecological condition of the Elbe Estuary.

Voxel-based analysis of cerebral glucose metabolism in mono- and dizygotic twins discordant for Alzheimer disease.

BACKGROUND: Sporadic Alzheimer disease (AD) is a multifactorial disease to which both genetic and environmental factors contribute. Therefore, twin pairs are useful in studying its pathogenesis and aetiology. Cerebral glucose metabolism has been found to be reduced in AD patients. METHODS: Cerebral glucose metabolism was studied in seven monozygotic (MZ) and nine same-sexed dizygotic (DZ) twin pairs discordant for AD using positron emission tomography. To obtain objective and explorative results concerning differences in glucose metabolism, the analysis was performed utilising modern voxel-based analysis methodology statistical parametric mapping and automated region-of-interest analysis. RESULTS: In the demented MZ and DZ co-twins, cerebral glucose metabolism was extensively reduced compared with controls. The non-demented MZ co-twins showed reduced metabolism in inferior frontal, lateral temporal, parietal and medial temporal cortices as well as in the thalamus, putamen and right amygdala. In contrast, no reductions were found in the non-demented DZ co-twins. The reduction found in the non-demented MZ co-twins may be an indicator of genetic susceptibility to AD.

Molybdopterin adenine dinucleotide and molybdopterin hypoxanthine dinucleotide in formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum (Marburg).

Formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum was purified to apparent homogeneity and found to contain per mol (apparent molecular mass 110 kDa) 0.6 mol molybdenum, 4 mol non-heme iron, 4 mol acid-labile sulfur, and in addition, 0.7 mol of a pterin-containing co-factor (apparent molecular mass 800 Da) which has been characterized. The pterin material was extracted after alkylation by iodoacetamide and the extract subjected to HPLC on Lichrospher 100 RP-18. Three pterin compounds were resolved. On the basis of their UV/visible spectra and of the products formed after cleavage by nucleotide pyrophosphatase and alkaline phosphatase they were identified as the [di(carboxamidomethyl)]-derivatives of molybdopterin guanine dinucleotide (MGD), of molybdopterin adenine dinucleotide (MAD), and of molybdopterin hypoxanthine dinucleotide (MHD). The three pterin dinucleotides were present in the proportions 1:0.4:0.1.

The molybdenum cofactor of formylmethanofuran dehydrogenase from Methanosarcina barkeri is a molybdopterin guanine dinucleotide.

The molybdenum cofactor of formylmethanofuran dehydrogenase from methanol-grown Methanosarcina barkeri was isolated as the [di(carboxamidomethyl)]-derivative. The alkylated factor showed an absorption spectrum and chemical properties identical to those recently reported for the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides. By treatment with nucleotide pyrophosphatase the factor was resolved into two components, which were identified as [di(carboxamidomethyl)]-molybdopterin and GMP by their absorption spectra, their retention times on Lichrospher RP-18, and by their conversion to dephospho-[di(carboxamidomethyl)]-molybdopterin and guanosine, respectively, in the presence of alkaline phosphatase. The GMP-moiety was sensitive to periodate, identifying it as the 5'-isomer. These results demonstrate that the molybdenum cofactor isolated from formylmethanofuran dehydrogenase contains the phosphoric anhydride of molybdopterin and 5'-GMP.

N-furfurylformamide as a pseudo-substrate for formylmethanofuran converting enzymes from methanogenic bacteria.

Methanofuran (4-[N-(4,5,7-tricarboxyheptanoyl-gamma-L-glutamyl)-gamma-L- glutamyl)-p-(beta-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan is a coenzyme involved in methanogenesis. The N-formyl derivative is an intermediate in the reduction of CO2 to CH4 and the disproportionation of methanol to CO2 and CH4. Formylmethanofuran dehydrogenase and formylmethanofuran:tetrahydromethanopterin formyltransferase are the enzymes catalyzing its conversions. We report here that the two enzymes from Methanosarcina barkeri and the formyltransferase from Methanobacterium thermoautotrophicum can also use N-furfurylformamide as a pseudo-substrate albeit with higher apparent Km and lower apparent Vmax values. N-Methylformamide, formamide, and formate were not converted indicating that the furfurylamine moiety of methanofuran is the minimum structure required for the correct binding of the coenzyme.

Formylmethanofuran dehydrogenase from methanogenic bacteria, a molybdoenzyme.

Formylmethanofuran dehydrogenase, a key enzyme of methanogenesis, was purified 100-fold from methanol grown Methanosarcina barkeri to apparent homogeneity and a specific activity of 34 mumol.min-1.mg protein-1. Molybdenum was found to co-migrate with the enzyme activity. The molybdenum content of purified preparations was 3-4 nmol per mg protein equal to 0.6-0.8 mol molybdenum per mol enzyme of apparent molecular mass 200 kDa. Evidence is presented that also formylmethanofuran dehydrogenase from H2/CO2 grown Methanobacterium thermoautotrophicum (strain Marburg) is a molybdoenzyme.

Cortical activation related to auditory semantic matching of concrete versus abstract words.

OBJECTIVE: The aim of this study was to examine cortical correlates of semantic memory processes in the auditory stimulus modality. METHODS: Event-related desynchronization (ERD) and synchronization (ERS) of the lower (8-10 Hz) and upper (10-12 Hz) alpha frequency bands of background EEG were studied in 10 subjects performing an auditory semantic matching task. The stimuli were abstract and concrete nouns which were presented sequentially in pairs. The task was to decide whether the two nouns belonged to the same semantic category or not. RESULTS: The presentation of the first stimulus (encoding) elicited ERS whereas the presentation of the second stimulus (semantic matching) elicited ERD. Abstract nouns presented as the second stimulus elicited ERD which was most prominent in the lower alpha frequency band, whereas the presentation of a concrete noun as the first stimulus elicited ERS, most prominently in the upper alpha frequency band. CONCLUSIONS: The auditorily elicited ERD/ERS reflects cortical activity associated with cognitive functions. The present findings demonstrate that the auditorily elicited ERD/ERS can reveal subtle differences in auditory information processing. Semantic memory processes (encoding and comparison) are reflected as varying responses in the two alpha frequency bands.

Follow-up of [11C]PIB uptake and brain volume in patients with Alzheimer disease and controls.

OBJECTIVE: In Alzheimer disease (AD), the accumulation pattern of beta-amyloid over time and its relationship with dementia severity are unclear. We investigated the brain uptake of the amyloid ligand (11)C-labeled Pittsburgh compound B ([(11)C]PIB) and volumetric brain changes over a 2-year follow-up in patients with AD and in aged healthy controls. METHODS: Fourteen patients with AD (mean age 72 years, SD 6.6) and 13 healthy controls (mean age 68 years, SD 5.4) were examined at baseline and after 2 years (patients with AD: mean 2.0 years, SD 0.2; controls: mean 2.1 years, SD 0.6) with [(11)C]PIB PET, MRI, and neuropsychological assessments. [(11)C]PIB uptake was analyzed with a voxel-based statistical method (SPM), and quantitative data were obtained with automated region-of-interest analysis. MRI data were analyzed with voxel-wise tensor-based morphometry. RESULTS: The [(11)C]PIB uptake of the patients with AD did not increase significantly during follow-up when compared with that of the controls. MRI showed progressive brain volume change in the patients with AD, e.g., in the hippocampal region, temporal cortex, and precuneus (p < 0.05). The mean Mini-Mental State Examination score of the patients with AD declined from 24.3 (SD 3.1) at baseline to 21.6 (SD 3.9) at follow-up (p = 0.009). Cognitive decline was also evident in other neuropsychological test results. Baseline neocortical [(11)C]PIB uptake ratios predicted subsequent volumetric brain changes in the controls (r = 0.725, p = 0.005). CONCLUSIONS: The results suggest no (or only little) increase in (11)C-labeled Pittsburgh compound B ([(11)C]PIB) uptake during 2 years of Alzheimer disease progression, despite advancing brain atrophy and declining cognitive performance. Nevertheless, changes in [(11)C]PIB uptake during a longer follow-up cannot be excluded. High cortical [(11)C]PIB uptake may predict ongoing brain atrophy in cognitively normal individuals.

CERAD test performances in amnestic mild cognitive impairment and Alzheimer's disease.

OBJECTIVES: The aim of the study was to examine the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) test performances cross-sectionally in patients suffering from amnestic mild cognitive impairment (MCI) and mild Alzheimer's disease (AD). Moreover, we wanted to determine the sensitivity to amnestic MCI and mild AD, as well as the specificity of different CERAD subtests in our study groups. MATERIAL AND METHODS: Fifteen healthy elderly individuals, 15 amnestic MCI patients and 15 probable AD patients suffering from mild dementia were tested with the CERAD neurocognitive dementia screening test. RESULTS: Significant differences were found in all CERAD tests except Constructional praxis (copy) and Clock drawing between the controls and the AD group. The MCI group was differentiated from the controls only in the Wordlist learning test. In the language tests the sensitivity to MCI and AD was quite low and the specificity very high. In the savings scores the sensitivity to AD was high, but the specificity rather low. The Wordlist recognition test screened no false positives using the current cut-off score and the sensitivity to AD was 0.6, but only one MCI patient was detected using the current cut-off score. Raising the cut-off score also raised the sensitivity to MCI without dramatic loss of specificity. Cut-off scores for the Wordlist learning test and Wordlist delayed recall, which have been found to differentiate normal aging from dementia, are lacking in the Finnish CERAD. The current data indicates that the Wordlist learning test might be relatively sensitive to MCI. CONCLUSIONS: The results indicate that the Finnish CERAD test battery with its current cut-off scores has low sensitivity to MCI, and using it as a sole cognitive screening instrument for MCI and preclinical dementia might result in false negatives.

Age, education and test performance on the Finnish CERAD.

OBJECTIVES: The purpose of this study was to clarify the effects of age and education on CERAD (Consortium to Establish a Registry for Alzheimer's Disease) test performances. MATERIAL AND METHODS: We present preliminary data on CERAD test results for a sample of elderly normal Finns (n = 40, age 58-85 years). RESULTS: Our results show no significant correlation between age and CERAD test results. Significant differences were found between subjects with low versus high education in nine CERAD test scores. The mean scores in the low-education group were near the cut-off point in the naming test, whereas the mean scores of the high-education group nearly reached the ceiling on six test scores. These differences were evident in the wordlist learning test, which has previously been argued to be unaffected by educational level. Savings scores were unaffected by education. CONCLUSION: The results underline the importance of taking into account the educational level of persons with subjective memory complaints when screening with CERAD for mild cognitive impairment and preclinical Alzheimer's disease.

Ischemic heart disease. Precarious coronary lesions and prognosis.

Coronary arteriograms in 113 patients whose cases were followed for 2 to 12 years were analyzed in an attempt to discover why in some patients with angina pectoris there are long stable clinical courses and in others the courses proceed rapidly to death. It was found that patients with completed coronary occlusions, usually with distal portions reconstituted by collateral, had favorable prognoses with prolonged longevity. Patients with multiple high grade narrowings of the coronary arteries, on the other hand, had poor prognoses with high attrition rates. The more stenotic lesions present in the coronary arteries, the higher the attrition rates. Completed coronary occlusions, therefore, have been classified as nonprecarious while local coronary stenoses greater than 50 percent of the lumen diameter have been classified as degrees of precariousness according to the number of lesions present. When compared with classification by number of diseased vessels and by arteriographic score of Friesinger, the nonprecarious cases had better prognoses than the precarious.

The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor.

Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6-carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes. A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5-0.8 mol molybdenum, 0.6-0.9 mol pterin, 28 +/- 2 mol non-heme iron and 28 +/- 2 mol acid-labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 mumol.min-1.mg-1 (kcat = 640 s-1) assayed with methylviologen (app. Km = 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.