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PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗
PubMed1975

[Effect of xylitol on increased intracranial pressure].

Increased intracranial pressure could be lowered by means of 250 ml 40% xylite (1,2,3,4,5-pentanpentol) in 17 patients, the average dosage amounted to 1.36 mg/kg, the lowering of the pressure to 54.4 plus or minus 18.4%. The effect lasted 109.5 plus or minus 35.4 min; the maximum occurring after 50.4 plus or minus 14.8 min. With the exception of 3 patients who complained of slight gastrointestinal disturbances, no side reactions were observed. Paralleling the lowering of CSF-pressure increased diuresis occurred amounting to 400% (including the infusion volume). The maxima of diuresis and lowering of CSF-pressure concur. Simultaneously we can see a highly significant lowering of urine potassium and a slightly significant lowering of sodium within the first 2 h, which after 3 h is clearly receding. There was no significant change in the values of serum electrolytes, bilirubin and transaminases (SGOT and SGPT); the residual urea was just a little lowered. The lowering of CSF-pressure after xylite surpasses the effect of the same dosage of sorbit and fructose.

Adult↗
PubMed1995

Chlorophyll a and carotenoid triplet states in light-harvesting complex II of higher plants.

Laser-flash-induced transient absorption measurements were performed on trimeric light-harvesting complex II to study carotenoid (Car) and chlorophyll (Chl) triplet states as a function of temperature. In these complexes efficient transfer of triplets from Chl to Car occurs as a protection mechanism against singlet oxygen formation. It appears that at room temperature all triplets are being transferred from Chl to Car; at lower temperatures (77 K and below) the transfer is less efficient and chlorophyll triplets can be observed. In the presence of oxygen at room temperature the Car triplets are partly quenched by oxygen and two different Car triplet spectral species can be distinguished because of a difference in quenching rate. One of these spectral species is replaced by another one upon cooling to 4 Ki demonstrating that at least three carotenoids are in close contact with chlorophylls. The triplet minus singlet absorption (T-S) spectra show maxima at 504-506 nm and 517-523 nm, respectively. In the Chl Qy region absorption changes can be observed that are caused by Car triplets. The T-S spectra in the Chl region show an interesting temperature dependence which indicates that various Car's are in contact with different Chl a molecules. The results are discussed in terms of the crystal structure of light-harvesting complex II.

Aerobiosis↗
PubMed1975

Production of molybdenum-coordinating compound by Bacillus thuringiensis.

Bacillus thuringiensis (ATCC 10792) produces a molybdenum reactive compound (given the trivial name chelin) during growth on iron-deficient medium. This compound accumulates in the culture medium in direct relation to the amount of L-arginine added and reaches a maximum concentration 24 to 48 h after the stationary phase of growth. Chelin absorbs light in the ultraviolet region with absorption maxima at 315 and 248 nm and minima at 284 and 240 nm. Chelin reacts with Na2MoO4, but not with Mo2O4(H2O)6-2+, to form a bright yellow molybdo-chelin complex which absorbs light with an absorption maximum at 330 nm, a minimum at 288 nm, and shoulders at 255 and 400 nm. The differential absorption of molybdo-chelin versus chelin at 425 nm can be used to quantify chelin. This differential absorbance is linear with increasing concentrations of Na2MoO4 and was used to calculate the molar extinction coefficient of molybdochelin at 425 nm (epsilon similar to 6,200). Chelin binds MoO4-2 minus to form a complex (molybdochelin) which migrates as a single band and elutes as a single peak, during acrylamide gel electrophoresis and Sephadex G-15 gel filtration. Molecular weight determinations using Sephadex G-15 gel filtration resulted in an estimated molecular weight of 550 for chelin and an estimated molecular weight of 760 for molybdo-chelin. The peptide nature of chelin is indicated by its positive ninhydrin reaction on thin-layer chromatography plates and by the presence of amino acids in acid-hydrolyzed samples. The major amino acid residues detected were threonine, glycine, and alanine.

Amino Acids↗
PubMed1990

Potentiometric and spectroscopic properties of the cytochrome o complex of Escherichia coli.

Cytochrome o purified from cell membranes of Escherichia coli shows two potentiometrically distinct species with midpoint oxidation-reduction potentials of +265 +/- 5 and +140 +/- 15 mV. The component with the higher potential reacted with carbon monoxide and so likely is the oxygen-reacting heme of the cytochrome o complex. It appears to be responsible for the absorption maximum at 564 nm in reduced minus oxidized difference spectra measured at 77 K. The midpoint potential of the other component was sensitive to oxidation by ferricyanide. This latter component had an absorption maximum at about 554 nm. The inhibitor 2-heptyl-4-hydroxyquinoline N-oxide inhibited reoxidation of reduced cytochrome o by oxygen and modified the spectroscopic behaviour of the 564 nm component. The ratio of the heights of the maxima in the alpha-band region of the absorption spectrum differed in cytochrome o prepared from cloned material from that found in cytochrome o from noncloned sources, in spite of the similar polypeptide compositions of the two preparations.

Carbon Monoxide↗
PubMed1986

Haemoprotein b-590 (Escherichia coli), a reducible catalase and peroxidase: evidence for its close relationship to hydroperoxidase I and a 'cytochrome a1b' preparation.

A reducible hydroperoxidase, haemoprotein b-590, has been purified 16-fold from a soluble fraction of Escherichia coli K12, grown anaerobically with glycerol and fumarate. The Mr of the native protein, determined by gel filtration, was 331,000 although a minor, smaller species with a Mr of 188,000 was also detected; both had catalase activities. Based on the subunit Mr, determined from SDS gel electrophoresis to be 75,000, the above species are tentatively identified as tetramers and dimers, respectively. The isoelectric point of both species was 4.4. The absorption spectrum of the isolated haemoprotein is typical of ferric, high-spin haem. The A405/A280 ratio never exceeded 0.27, a value half of that obtained for E. coli hydroperoxidase I. On reduction with dithionite, the gamma, beta, and alpha bands were at 441, 559 and 590 nm respectively, the alpha-band being unusually distinct. Treatment of the reduced form with CO gave a sharp prominent gamma-band at 426 nm and caused significant shifts of the alpha and beta bands to shorter (574 and 545 nm) wavelengths. The pyridine haemochrome spectra showed the haem to be protohaem IX; the spectra were featureless between 580 and 630 nm, thus excluding the presence of haem a. However, some features of the difference spectra of the haemoprotein were reminiscent of cytochrome a1, notably the maxima in reduced minus oxidized spectra at 444 and 593 nm and the peaks and troughs in CO difference spectra at 426 and 446 nm respectively. The haemoprotein had high catalase activity: Vmax was 2.3 X 10(6) mol H2O2 (mol haem)-1 min-1 and the Km was 11 mM. At 10 mM-H2O2 the first order rate constant was 0.3 X 10(7) M-1 s-1. The haemoprotein was also a peroxidase with o-dianisidine or 2,3',6-trichloroindophenol as substrates; for the latter substrate, the Km was 0.18 mM. It is concluded that haemoprotein b-590 strongly resembles the hydroperoxidase I purified by Claiborne & Fridovich (Journal of Biological Chemistry 254, 4245-4252, 1979) and that a similar haemoprotein was mistaken for a cytochrome a1 b complex by Barrett & Sinclair (Abstracts of the 7th International Congress of Biochemistry, Tokyo, H-107, p. 907, 1967).

Catalase↗
PubMed1993

Structure of the complex between pyridoxal 5'-phosphate and the tyrosine 225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase determined by isotope-edited classical Raman difference spectroscopy.

The azomethine (Schiff base) linkage between the epsilon-amino group of active-site lysine 258 and the carbonyl moiety of enzyme-bound pyridoxal 5'-phosphate (PLP) normally exhibits absorbance maxima at ca. 360 (high-pH form) or ca. 430 nm (low-pH form). However, the absorbance maximum is shifted from 358 to 386 nm, a value which is similar to that of free PLP (lambda max = 388 nm), in a mutant form of Escherichia coli aspartate aminotransferase (AATase) in which tyrosine 225, which normally donates a hydrogen bond to the phenolate function of PLP, has been replaced with phenylalanine (Y225F). This spectral shift suggested that PLP binds to Y225F as the free aldehyde. The following evidence from isotope-edited classical Raman spectroscopy proves conclusively that the near-UV spectrum is anomalous and that PLP is bound to Y225F as a Schiff base: (1) A strong cofactor peak at 1630 cm-1 in the holoenzyme-minus-apoenzyme difference spectrum of the unprotonated form of Y225F is red-shifted by 18 cm-1 in enzyme labeled with 15N at lysine 258 and other positions. (2) This isotope-induced red shift is similar to that observed in the unprotonated form of the model Schiff base, PLP-valine. (3) The Raman spectrum of Y225F is unchanged in H(2)18O, while peaks at ca. 1670 cm-1 in the spectrum of free PLP or in that of a mutant of AATase in which Lys-258 is replaced with Ala, are red-shifted by ca. 30 cm-1 in H(2)18O.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Aminotransferases↗
PubMed1989

Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr.

In eubacteria, the tRNA transglycosylase (Tgt) in specific tRNAs exchanges a guanine in the anticodon for 7-aminomethyl-7-deazaguanine, which is finally converted to queuosine. The tgt gene of Escherichia coli has been mapped at 9 min on the genome, and mutant pairs containing an intact or mutated tgt allele were obtained after transduction of the tgt locus by P1 bacteriophages into a genetically defined E. coli strain (S. Noguchi, Y. Nishimura, Y. Hirota, and S. Nishimura, J. Biol. Chem. 257:6544-6550, 1982). These tgt mutants grew anerobically with fumarate as an electron acceptor, while nitrate or trimethylamine N-oxide could not be reduced. Furthermore, molybdate reductase activity was almost lacking and the characteristic absorption maxima, corresponding to cytochrome a1 and the cytochrome d complex, were not detectable in low-temperature reduced-minus-oxidized difference spectra in anaerobically grown cells. Transduction of the mutated tgt locus into another E. coli recipient resulted in tgt mutants without anaerobic defects. Transformation of the original tgt mutants with an fnr gene-containing plasmid reversed the anaerobic defects. Clearly, the original tgt mutants harbor a second mutation, affecting the anaerobic regulator protein Fnr. The results suggest that fnr is involved in anaerobic control of components of the cytochrome d complex and of the redox system that transfers electrons to molybdate. F' plasmids containing a fused lacI-lacZ gene with the nonsense codon UAG at different positions in the lacI part were transferred to E. coli strains with a mutated or nonmutated tgt locus but intact in fnr. A twofold increase in the frequency of incorrect readthrough of the UAG codon, dependent on the codon context, was observed in the tgt mutant and is suggested to be caused by a tRNA(Tyr) with G in place of queuosine.

Base Sequence↗
PubMed1987

Cytochrome pools in membranes of Escherichia coli grown aerobically on L-proline.

The cytochromes of membranes of the cydA mutant Escherichia coli GR19N grown on a proline-amino acid medium were examined. Reduced minus oxidized difference spectra (including fourth-order finite difference spectra) showed that cytochromes with absorption maxima at 554-555, 556-557, 560-561.5 and 563.5-564.5 nm were present. In addition, there were two components with absorption maxima at 548.5 and 551.5 nm which made a minor contribution to the alpha-band absorbance. These were not examined further. Two pools within the cytochromes were detected. One pool, which was reduced rapidly by the substrates NADH, formate and succinate, consisted of cytochromes of the cytochrome o complex. These cytochromes had absorption maxima at 555, 557 and 563.5 nm. In addition, the low-potential cytochrome associated with formate dehydrogenase was reduced rapidly by formate, and a component absorbing at 560-561.5 nm was also present in this pool. The second pool of cytochromes was reduced more slowly by substrate, although the rate was accelerated greatly in the presence of the electron mediator phenazine methosulfate. These cytochromes absorbed maximally at about 556.5 nm. A portion of the cytochrome in this pool was reoxidized by fumarate. This cytochrome may be a component of the fumarate reductase pathway, since the membranes showed high NADH-fumarate reductase activity. The respiratory chain inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide appeared to act at two sites. One site of inhibition was between the dehydrogenases and the cytochromes. A second site of inhibition was located in the cytochrome o complex between cytochrome b-564 and oxygen.

Aerobiosis↗
PubMed1992

Difference spectroscopic characterisation of the cytochrome complement in Acanthocheilonema viteae.

(Dithionite-reduced) minus (ferricyanide-oxidised) difference spectra of 600 x g and 12,000 x g subcellular pellet fractions of adult male Acanthocheilonema viteae exhibited alpha-absorption maxima (296 K) attributable to Cyt c555, Cyt b562 and aa3 (600-605 nm). The gamma(Soret) maximum of both fractions was evident at 427 nm, with a shoulder at 432-434 nm. 600 x g and 12,000 x g pellet fractions of adult female and mixed-sex adult A. viteae exhibited similar absorption maxima. (Succinate-reduced)--(ferricyanide-oxidised) difference spectra of the 12,000 x g pellet fraction of mixed-sex adult A. viteae showed absorption maxima at 555 and 562 nm, 600 and 630 nm, suggesting the reduction of Cyt c555, Cyt b562, Cyt aa3 (600 nm) and an unidentified species (630 nm peak) Antimycin A (10(-6) M) induced the disappearance of the maxima at 555, 600 and 630 nm corresponding to Cyt c555, Cyt aa3 and the unidentified species; the maximum at 562 nm prevailed in the presence of antimycin A. These antimycin A induced changes can be cited as classical evidence for the functional involvement of these a, b and c type cytochromes in respiratory electron transport. (Dithionite reduced + CO)--(dithionite reduced) difference spectra suggest that adult A. viteae may have one or more CO-binding-species, one of which appears to be a low-spin-haemoprotein with a b-type or c-type haem, which has essentially an electron carrier function rather than a ligand binding function.

Animals↗
PubMed1996

A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system.

The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant (Robillard et al., 1996). The steady-state fluorescence spectra of EIImtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I- quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I-. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 384 or by a tyrosinate at position 458 which is nearby in the folded protein (AB et al., in preparation; van Montfort et al., in preparation). The observed emission maxima and accessibilities of the tryptophans at the different positions are consistent with the predicted topology of the enzyme (Sugiyama et al., 1991). When mannitol is bound to wild-type EIImtl, an increase in fluorescence emission intensity was observed (Wood, 1988) which can now be attributed primarily to increased fluorescence intensity of the tryptophan at position 30.

Amino Acid Sequence↗
PubMed1994

[Postoperative warming therapy in the recovery room. A comparison of radiative and convective warmers].

Hypothermia (Tcore < 36 degrees C) can be observed in 60%-80% of all admissions to the post-anaesthetic recovery unit. Effective warming devices may accelerate rewarming, improve patient comfort, and suppress shivering thermogenesis. This study was designed to compare the efficiency of warming devices in extubated postoperative patients and their effect on postoperative oxygen uptake (VO2). METHODS. Thirty-five ASA I and II patients after laparoscopic hernioplastic repair with core temperatures < 36 degrees C were randomly assigned to either postoperative nursing under a radiant heater (group R, n = 11, Aragona Thermal Ceilings CTC X, Aragona Medical AB, Täby, Sweden), a forced air system (group L, n = 12, Bair Hugger, Augustine Medical Inc., Eden Prairie, Minnesota, USA), or a normal cotton hospital blanket (group K, n = 12). Anaesthesia was conducted totally intravenously with propofol, alfentanil, and vecuronium. Mean body temperature and total body heat were calculated from urinary bladder temperature and four subcutaneous temperature measurements. The rate of thermogenesis was calculated from continuous measurement of VO2 (Datex Deltatrac Metabolic Monitor, Datex Instrumentarium Corp., Helsinki, Finland). Heat balance was derived from the increase in total body heat minus body heat production. Heart rate and noninvasive blood pressure were measured by the Cardiocap (Datex Instrumentarium Corp., Helsinki, Finland). All data were transferred to an IBM-compatible computer at 60-s intervals. Measurements were stopped when core temperature reached 37 degrees C. The rate of change was calculated for each variable for the period 15 min after the beginning of rewarming to attainment of 37 degrees C. Data are presented as median, minima, and maxima (min<==>max); the Mann-Whitney U test was used to test for significance of group differences. RESULTS. All groups were comparable for body weight, height, age, and amount of postoperative infusions. Temperatures at admission were 35.2 (33.4<==>35.9), 34.7 (34.3<==>35.8), and 35.4 (34.3<==>35.9) degrees C for groups R, B, and K, respectively. No significant differences in the rate of central rewarming could be found for these groups with 0.81 (0.41<==>1.32), 0.76 (0.40<==>1.07), and 0.70 (0.37<==>1.13) degrees C/h (Fig. 1). The mean VO2 of 3.41 (3.07<==>3.73), 3.55 (2.78<==>4.06), and 3.79 (2.51<==>7.00) ml/kg/min also did not differ significantly (Fig. 3). Significant differences between groups R and B [4.39 (3.74<==>6.19) and 4.30 (3.46<==>6.67) ml/kg/min] and K [5.92 (3.79<==>10.64) ml/kg/min] were found for VO2 maxima during the course of investigation (Fig. 4). The heat balance revealed significant differences among treatment and control groups with -88 (-226<==>+30), -41 (-212<==>+12), and -191 (-265<==>-86) kJ/h for groups R, B, and K. We additionally calculated the heat balance as a quotient, which showed 0.70 (0.22<==>1.07), 0.86 (0.44<==>1.04), and 0.49 (0.31<==>0.79) for groups R, B, and K (Fig. 4). The mean rate-pressure product of all groups did not differ significantly during the period of investigation. CONCLUSIONS. Neither external heat supply by radiant heat nor by a forced warm air system significantly reduced rewarming time in extubated, awake patients. As measured by heat balance, both active treatments saved about 20% more body heat production than in the control group. Continuing peripheral vasoconstriction may be the reason for the low efficiency of heat transfer. Thermal treatment did reduce the peak load (max. VO2) on the oxygen transport systems, though shivering was treated by pethidine if it occurred. External rewarming did not reduce the average load (mean VO2). Thus, concerning the goal of accelerating rewarming, it appears more rational to prevent intraoperative heat loss. For a comparison of efficiency of different warming devices, postoperative extubated patients do not appear to be an ideal model for study.

Adult↗