Incorporation of 4-amino-5-hydroxymethylpyrimidine into thiamine by microorganisms.

One possible route for the biosynthesis of the (4-amino-2-methyl-5-pyrimidyl)-methyl moiety of thiamine would involve the formation of a methyl group on the demethylated pyrimidine, 4-amino-5-hydroxymethylpyrimidine, before its incorporation into thiamine. This possibility was tested by preparing the 4-amino-5-hydroxymethylpyrimidine and feeding it to Escherichia coli. Bacillus subtilis, and Saccharomyces cerevisiae. Analysis of the thiamine produced by these organisms showed that 4-amino-5-hydroxymethylpyrimidine was readily incorporated into thiamine without the addition of a methyl group, and no evidence was found for the conversion of this pyrimidine into normal methylated pyrimidine. Substitution of the demethylated thiamine for thiamine had no effect on the growth rate or the yield of E. coli cells. Complete substitution of the thiamine with the (4-amino-5-pyrimidyl)-methyl moiety was possible in an E. coli pur I mutant. The extent of incorporation of the demethylated pyrimidine decreased in some organisms and increased in others by the addition of adenine to the growth medium; this difference led to a simple test to separate organisms that use 5-aminoimidazole ribonucleotide for the biosynthesis of thiamine pyrimidine from those that do not.

Occurrence of S-methyl thioesters in urines of humans after they have eaten asparagus.

Gas chromatography-mass spectrometry was used to determine the odor-causing agent (or agents) present in the urines of humans after they have eaten asparagus. S-Methyl thioacrylate and S-methyl 3-(methylthio)thiopropionate were identified from methylene chloride extracts of such urines and appear to be the odor-causing compounds. Methanethiol, the previously reported odor-causing agent, was not detected in these methylene chloride extracts.

Inositol isomers: occurrence in marine sediments.

A combined gas chromatographic-mass spectrometric technique was used to identify and quantitate the occurrence of myo-, chiro-, and scyllo-inositol in marine sediments. The most abundant isomer was myo-inositol. These inositols were found in all the organic-rich sediment samples examined, and the amount of inositol decreased steadily with the age of the sample. A small fraction of the inositols occurred as hexaphosphate esters.

Vitamin A deficiency: effect on mosquito eye ultrastructure.

Mosquitoes (Aedes aegypti) were reared aseptically for one generation on an artificial diet containing neither vitamin A nor its usual precursor in animals, betacarotene. Function (electrical response to light) in the compound eyes of these animals was severely impaired. Ultrastructure of the photoreceptor cells was abnormal in two respects: multivesicular bodies were absent, and masses of smooth membrane lamellae were present near the nucleus. The organization of the photoreceptor organelle, the rhabdomere, was normal. The eyes of control mosquitoes, to whose diet beta-carotene was added, were functionally and structurally normal. Multivesicular bodies were normally abundant and the perinuclear membrane masses were not present.

Structure of an amide bond forming F(420):gamma-glutamyl ligase from Archaeoglobus fulgidus -- a member of a new family of non-ribosomal peptide synthases.

F(420) is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F(420)-dependent photolysis in DNA repair. Coenzyme F(420)-2 biosynthesis from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and lactaldehyde involves six enzymatic steps and five proteins (CofA, CofB, CofC, CofD, and CofE). CofE, a F(420)-0:gamma-glutamyl ligase, is responsible for the last two enzymatic steps; it catalyses the GTP-dependent addition of two L-glutamate residues to F(420)-0 to form F(420)-2. CofE is found in archaea, the aerobic actinomycetes, and cyanobacteria. Here, we report the first crystal structure of the apo-F(420)-0:gamma-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 A and 1.35 A resolution, respectively. The structure of CofE-AF reveals a novel protein fold with an intertwined, butterfly-like dimer formed by two-domain monomers. GDP and Mn(2+) are bound within the putative active site in a large groove at the dimer interface. We show that the enzyme adds a glutamate residue to both F(420)-0 and F(420)-1 in two distinct steps. CofE represents the first member of a new structural family of non-ribosomal peptide synthases.

High risk for venous thromboembolism in diabetics with hyperosmolar state: comparison with other acute medical illnesses.

BACKGROUND: Diabetes mellitus is generally not recognized as an important risk factor for venous thromboembolism (VTE). However, clinical observations and case reports have suggested that patients with diabetes and hyperosmolarity may be at increased risk for VTE. OBJECTIVES: To determine the risk of VTE in patients hospitalized for diabetes with hyperosmolar state compared to patients with other acute medical illnesses. PATIENTS/METHODS: The California Patient Discharge Data Set was used to determine the incidence of first-time VTE in all patients admitted between 1995 and 2000 for diabetes with hyperosmolarity and 11 other acute medical conditions. Proportional hazard modeling was used to adjust for age, race, gender, and prior hospitalization within 3 months. RESULTS: Among 2859 patients with diabetes and hyperosmolarity, 34 (1.2%) developed VTE during the hospitalization and 14 (0.5%) developed VTE within 91 days after discharge. In an adjusted multivariate model comparing the risk of VTE to cases with depression, patients with hyperosmolarity had a significantly higher risk of VTE [hazard ratio (HR) = 16.3; 95% confidence interval (CI): 10-25] comparable to the risk associated with sepsis (HR = 19.3; 95% CI: 13-29) or acute connective tissue disease (HR = 21; 95% CI: 15-31). Compared to uncomplicated diabetes, patients with hyperosmolarity had a significantly higher risk of VTE (HR = 3.0; 95% CI: 2.1-4.5) whereas patients with ketoacidosis were not at higher risk (HR = 1.2; 95% CI: 0.8-1.7). CONCLUSIONS: Patients hospitalized for diabetes with hyperosmolarity are at increased risk for developing VTE both during their inpatient stay and in the 3 months after discharge. Thromboprophylaxis in these patients appears warranted, and extended prophylaxis for after hospital discharge should be studied.

Runoff water quality from turfgrass established using volume-based composted municipal biosolids applications.

Municipal programs for turfgrass establishment recommend large volume-based application rates of composted municipal biosolids (CMB). This study compared runoff water quality among combinations of two common turfgrass establishment practices and two CMB sources. Bryan- or Austin-CMB were incorporated into 5 cm of soil at a rate of 12.5 or 25% by volume (v/v) on an 8.5% slope. Tifway bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy, var. Tifway] sprigs were planted and established; sod, produced at a separate site using either CMB amendment at the 25% v/v rate, was transplanted to the runoff plots on the same day. A mature stand of bermudagrass was used as a control. Runoff water was collected after each of eight natural rain events during the sampling period. Total runoff water loss (mm) was similar for the CMB-amended sprigged and transplanted sod stands. The concentration of total dissolved P (TDP) in runoff water was greatest from the transplanted sod in the first seven rain events (4.1 to 7.5 mg L(-1)). The concentration of TDP in runoff water was similar at both the 12.5 and 25% v/v incorporation rates. Regression analysis indicated Mehlich-3-extractable soil test P concentrations in soil amended with CMB were positively correlated to concentration and mass loss of dissolved P in runoff. At similar application rates, dissolved P loss in runoff water was reduced by incorporating CMB into the soil on site rather than transplanting sod produced with CMB.

4-Hydroxybenzyl alcohol. A metabolite produced during the biosynthesis of thiamine in Escherichia coli.

4-Hydroxybenzyl alcohol was identified by gas chromatography-mass spectrometry as a metabolite of Escherichia coli when it is grown on a medium containing no thiamine or 4-methyl-5-beta-hydroxyethyl thiazole. 4-Hydroxybenzyl alcohol was found to be derived from L-tyrosine and the amount produced was found to be inhibited by the addition of thiamine to the growth medium. The amount of 4-hydroxybenzyl alcohol produced, as measured by isotopic dilution, was shown to be equivalent to the amount of thiamine formed. Based on these observations, it was concluded that 4-hydroxybenzyl alcohol is the cleavage product produced during the biosynthesis of the thiazole moiety of thiamine from tyrosine.

Methanopterin biosynthesis: methylation of the biosynthetic intermediates.

The basic scheme for the biosynthesis of methanopterin (MPT) in Methanobacterium thermoautotrophicum strain DeltaH, and M. thermoautotrophicum strain Marburg, has been shown to be the same as that recently determined for Methanosarcina thermophila strain TM-1. This scheme has, as one of its unique steps, the condensation of 4-aminobenzoic acid with 5-phospho-alpha-d-ribosyl diphosphate (PRPP) to form 4-(beta-d-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P). Labeling experiments with each of these organisms have established that the sites in the overall sequence of reactions from beta-RFA-P to MPT, where the S-adenosylmethionine-dependent C-9 and C-7 methylations of the pterin-containing intermediates occur, are organism related. In this work, cell extracts of M. thermoautotrophicum strain DeltaH, and M. thermoautotrophicum strain Marburg were found to contain significant amounts of methanopterin lacking the phosphate and 2-hydroxyglutaric acid groups.

Cholesterol beta-D-glucoside-6'-O-palmitate, a metabolite of Pythium sylvaticum.

Cholesterol beta-D-glucoside-6'-O-palmitate has been identified as a polar metabolite in the mycelium of mated cultures of Pythium sylvaticum grown in the presence of cholesterol. The structure was confirmed by synthesis of the metabolite. Similar steroid beta-D-glucoside-6'-O-palmitates were obtained from beta-sitosterol and campesterol when these sterols were added to cultures of P-sylvaticum. Corresponding esters of myristic and stearic acids were also detected.

The origin of the nitrogen atom in the thiazole ring of thiamine in Escherichia coli.

Methods are described for the isolation and gas chromatographic-mass spectrometric analysis of the 4-methyl-5-beta-hydroxyethyl thiazole moiety of thiamine in microbial cells. Using these methods, it was determined that in Escherichia coli the nitrogen atom in the thiazole ring of thiamine is derived solely from L-tyrosine.

The first examples of (S)-2-hydroxyacid dehydrogenases catalyzing the transfer of the pro-4S hydrogen of NADH are found in the archaea.

Reduction of 2-oxoacids to the corresponding (S)-2-hydroxyacids is an important transformation in biochemistry. To date all (S)-2-hydroxyacid dehydrogenases belonging to the L-lactate/L-malate dehydrogenase family have been found to transfer the pro-4R hydrogen of either NADH or NADPH to C-2 of the 2-oxoacid substrates during their reduction. Here, we report that recombinantly generated (S)-2-hydroxyacid dehydrogenases present in the methanoarchaea Methanococcus jannaschii and Methanothermus fervidus use the pro-4S hydrogen of NADH to reduce a series of 2-oxoacids to the corresponding (S)-2-hydroxyacids. This information as well as the low sequence identity between these archaeal enzymes and the L-lactate/L-malate family of enzymes indicate that these enzymes are not evolutionary related and therefore constitute a new class of (S)-2-hydroxyacid dehydrogenases.

Rhodopsin of the larval mosquito.

Larvae of the mosquito Aedes aegypti have a cluster of four ocelli on each side of the head. The visual pigment of each ocellus of mosquitoes reared in darkness was characterized by microspectrophotometry, and found to be the same. Larval mosquito rhodopsin (lambda(max) = 515 nm) upon short irradiation bleaches to a stable photoequilibrium with metarhodopsin (lambda(max) = 480 nm). On long irradiation of glutaraldehyde-fixed tissues or in the presence of potassium borohydride, bleaching goes further, and potassium borohydride reduces the product, retinal, to retinol (vitamin A(1)). In the presence of hydroxylamine, the rhodopsin bleaches rapidly, with conversion of the chromophore to retinaldehyde oxime (lambda(max) about 365 nm).

Diminution and enlargement of the mosquito rhabdom in light and darkness.

The rhabdoms of the larval ocelli of the mosquito Aedes aegypti undergo morphological light and dark adaptation over periods of hours. The rhabdom enlarges during dark adaptation and grows smaller during light adaptation. Diminution is exponential, enlargement linear, and rates of change are proportional to log light intensity. Rhabdoms maintained at a constant intensity level off at a constant volume proportional to log intensity. We argue that changes in rhabdom volume after changes in light intensity reflect an influence of light on the turnover of photoreceptro membrane, and that the volumes at which rhabdoms level off represent equilibria between opposed processes of membrane loss and renewal.

Rhodopsin and porphyropsin fields in the adult bullfrog retina.

Though it had been supposed earlier that the bullfrog undergoes a virtually complete metamorphosis of visual systems from vitamin A(2) and porphyropsin in the tadpole to vitamin A(1) and rhodopsin in the adult, the present observations show that the retina of the adult frog may contain as much as 30-40% porphyropsin, all of it segregated in the dorsal zone. The most dorsal quarter of the adult retina may contain 81-89% porphyropsin mixed with a minor amount of rhodopsin; the ventral half contains only rhodopsin. Further, the dorsal zone contains a two to three times higher concentration of visual pigments than the ventral retina. The pigment epithelium underlying the retina contains a corresponding distribution of vitamins A(1) and A(2), predominantly vitamin A(2) in the dorsal pigment epithelium, exclusively vitamin A(1) in the ventral zone. The retina accepts whatever vitamin A the pigment epithelium provides it with, and turns it into the corresponding visual pigment. Thus, a piece of light-adapted dorsal retina laid back on ventral pigment epithelium regenerates rhodopsin, whereas a piece of light-adapted ventral retina laid back on dorsal pigment epithelium regenerates predominantly porphyropsin. Vitamin A(2) must be made from vitamin A(1), by dehydrogenation at the 3,4-bond in the ring. This conversion must occur in the pigment epithelium, presumably through the action of a vitamin A-3,4-dehydrogenase. The essential change at metamorphosis is to make much less of this dehydrogenase, and to sequester it in the dorsal pigment epithelium. Some adult bullfrogs, perhaps characteristically taken in the summer, contain very little porphyropsin-only perhaps 5%-still sequestered in the dorsal retina. The gradient of light over the retinal surface has little if any effect on this distribution. The greater density of visual pigments in the dorsal retina, and perhaps also-although this is less clear-the presence of porphyropsin in this zone, has some ecological importance in increasing the retinal sensitivity to the dimmer and, on occasion, redder light received from below.

Spectral sensitivity of larval mosquito ocelli.

The spectral sensitivity of lateral ocelli in both wild-type and white-eyed larvae of the yellow fever mosquito Aedes aegypti L. (reared in darkness) was measured by means of the electroretinogram. The spectral sensitivity is maximal at about 520 nm, with a small secondary peak near 370 nm. When allowance is made for some screening and filtering by the eye tissues, the spectral sensitivity is in reasonable agreement with the absorption spectrum of ocellar rhodopsin (lambda(max) = 515 nm).

Accuracy and precision of a portable anticoagulation monitor in a clinical setting.

BACKGROUND: Office-based anticoagulation monitors offer significant advantages in convenience, yet their performance has been inadequately characterized. METHODS: We characterized the performance of a portable anticoagulation monitoring system with respect to precision and agreement with a reference laboratory. Eighty-five patients from a university outpatient anticoagulation clinic provided 143 whole blood sample pairs for evaluating agreement between the monitor and the laboratory. Fifty-four patients each provided a second pair of samples for assessing the monitor's precision, and 23 pairs of measurements from the reference laboratory were used for assessing the laboratory's precision. Anticoagulation was measured using International Normalized Ratio (INR) values. Agreement between monitor and laboratory was evaluated as the difference between paired measurements. Precision was calculated as the within-patient standard deviation based on paired values. RESULTS: Within the range of 2.0 to 3.0 INR units, the monitor yielded values that were up to 0.3 units higher on average than the laboratory values. Within the range of greater than 3.0 to 4.5 INR units, the monitor yielded values that were up to 0.5 units lower on average than the laboratory values. Seventy-five percent of paired monitor and laboratory values were within 0.7 INR units; 90% were within 0.9 units. Within-patient standard deviation was 0.23 units for the monitor and 0.19 units for the laboratory. CONCLUSIONS: The monitor differed systematically from the laboratory and was moderately less precise. The magnitude of these effects was not great, however, and accuracy was best at around INR = 3.0, the border between low and high therapeutic ranges. The clinic-based monitor is useful for patients requiring frequent surveillance of anticoagulation status.

Behçet's syndrome in a family with inflammatory bowel disease.

Although the gastrointestinal and systemic features of Behcet's syndrome and inflammatory bowel disease overlap to a considerable extent, they are generally viewed as two distinct diseases. We evaluated three members of a family who have inflammatory bowel lesions, two of whom met criteria for Behcet's syndrome. The propositus had classic features of both Crohn's ileocolitis and Behcet's syndrome. A daughter, who never met criteria for Behcet's syndrome, had undergone colectomy for ulcerative colitis. A second daughter had classic features of Behcet's syndrome, including recurrent episodes of colitis with distinct aphthous ulcers in the colon. The findings in this family suggest that inflammatory bowel disease and Behcet's syndrome may be closely related and part of a spectrum of disease rather than distinct disease entities.

Length of hospital stay for treatment of deep venous thrombosis and the incidence of recurrent thromboembolism.

BACKGROUND: Current guidelines suggest that all patients with acute deep venous thrombosis should be treated with intravenous heparin for at least 5 days, overlapping with warfarin sodium for 4 to 5 days. METHODS: Using linked state of California hospital discharge records from 1991 to 1994 we identified patients with acute deep venous thrombosis without pulmonary embolism, and determined the 6-month cumulative incidence of rehospitalization for recurrent thromboembolism. Coding was validated by reviewing the charts of 218 patients matched with the statewide data from 4 local hospitals. RESULTS: A total of 36924 linked records met study criteria. In the validation group, objectively confirmed thrombosis that was treated with intravenous heparin followed by warfarin was noted in 20%, 65%, 94%, and 95% of the patients who were hospitalized for 2 or fewer days or 3, 4, or 5 or more days, respectively. Statewide, among patients hospitalized for 3, 4, 5, and 6 days, the 6-month cumulative incidence of hospitalization for recurrent thromboembolism was 5.4%, 5.1%, 5.4%, and 6.0%, respectively. Multivariate modeling of patients hospitalized for 3 to 10 days revealed that recurrent thromboembolism was associated with the length of hospitalization (odds ratio [OR], 1.06 each additional day; 95% confidence interval [CI], 1.04-1.08), presence of malignancy (OR, 1.58; 95% CI, 1.46-1.68), age (OR, 0.85 each 10 years; 95% CI, 0.84-0.86), dementia (OR, 0.38; 95% CI, 0.26-0.49), hospitalization for multiple injuries within 3 months (OR, 0.46; 95% CI, 0.32-0.60), and surgery within 3 months (OR, 0.84; 95% CI, 0.78-0.90). CONCLUSIONS: We found no evidence that a stay of 4 days for treatment of deep venous thrombosis was associated with a higher rate of recurrent thromboembolism compared with hospitalization for 5 or more days. Although the evidence was not as strong, the incidence of recurrent thromboembolism after a stay of 3 days appeared comparable with that after a stay of 5 days. These findings suggest that fewer than 5 days of intravenous heparin overlapping with warfarin may provide effective initial treatment for deep venous thrombosis among patients deemed ready for hospital discharge.

Effect of weight, sex, age, clinical diagnosis, and thromboplastin reagent on steady-state intravenous heparin requirements.

BACKGROUND: There is a significant direct relationship between steady-state intravenous heparin dose requirements and total body weight. Less is known about whether sex, age, clinical diagnosis, and the thromboplastin used to measure the activated partial thromboplastin time (aPTT) affect heparin dose requirements. METHODS: Four cohorts of patients treated with intravenous heparin were gathered from 3 hospitals: 2 cohorts with deep vein thrombosis (DVT) and 2 cohorts with coronary artery disease (CAD). For each clinical diagnosis, half the patients were monitored using one aPTT reagent and the remainder were monitored using a second reagent. Heparin doses and aPTT measurements were recorded, and the dose necessary to achieve an aPTT ratio of 2.0 was calculated using a computer software program. RESULTS: We analyzed the records of 340 patients: 165 with DVT and 175 with CAD. Using analysis of variance, there was a significant difference in the steady-state heparin requirements among patients with DVT compared with patients with CAD (P < .001). For each clinical diagnosis, the use of a different thromboplastin reagent did not affect heparin dose requirements (P > .42). Linear regression modeling disclosed that the steady-state heparin dose for patients with DVT was a function of weight plus an effect modifier involving weight and age, whereas for patients with CAD there was only a weak relationship with weight. CONCLUSIONS: Steady-state heparin dose requirements were significantly different in patients with DVT compared with patients with CAD, suggesting that different dosing nomograms are needed for each condition. For patients with DVT, the accuracy of the initial heparin dose estimate may be improved by considering the patient's age and weight.