Association between levels of pentraxin 3 and incidence of chronic kidney disease in the elderly.

OBJECTIVE: Higher levels of the novel inflammatory marker pentraxin 3 (PTX3) predict cardiovascular mortality in patients with chronic kidney disease (CKD). Yet, whether PTX3 predicts worsening of kidney function has been less well studied. We therefore investigated the associations between PTX3 levels, kidney disease measures and CKD incidence. METHODS: Cross-sectional associations between serum PTX3 levels, urinary albumin/creatinine ratio (ACR) and cystatin C-estimated glomerular filtration rate (GFR) were assessed in two independent community-based cohorts of elderly subjects: the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS, n = 768, 51% women, mean age 75 years) and the Uppsala Longitudinal Study of Adult Men (ULSAM, n = 651, mean age 77 years). The longitudinal association between PTX3 level at baseline and incident CKD (GFR <60 mL(-1) min(-1) 1.73 m(-2) was also analysed (number of events/number at risk: PIVUS 229/746, ULSAM 206/315). RESULTS: PTX3 levels were inversely associated with GFR [PIVUS: B-coefficient per 1 SD increase -0.16, 95% confidence interval (CI) -0.23 to -0.10, P < 0.001; ULSAM: B-coefficient per 1 SD increase -0.09, 95% CI -0.16 to -0.01, P < 0.05], but not ACR, after adjusting for age, gender, C-reactive protein and prevalent cardiovascular disease in cross-sectional analyses. In longitudinal analyses, PTX3 levels predicted incident CKD after 5 years in both cohorts [PIVUS: multivariable odds ratio (OR) 1.21, 95% CI 1.01-1.45, P < 0.05; ULSAM: multivariable OR 1.37, 95% CI 1.07-1.77, P < 0.05]. CONCLUSIONS: Higher PTX3 levels are associated with lower GFR and independently predict incident CKD in elderly men and women. Our data confirm and extend previous evidence suggesting that inflammatory processes are activated in the early stages of CKD and drive impairment of kidney function. Circulating PTX3 appears to be a promising biomarker of kidney disease.

Hyperoxia affects the regional pulmonary ventilation/perfusion ratio: an electrical impedance tomography study.

BACKGROUND: The way in which hyperoxia affects pulmonary ventilation and perfusion is not fully understood. We investigated how an increase in oxygen partial pressure in healthy young volunteers affects pulmonary ventilation and perfusion measured by thoracic electrical impedance tomography (EIT). METHODS: Twelve semi-supine healthy male volunteers aged 21-36 years were studied while breathing room air and air-oxygen mixtures (FiO2) that resulted in predetermined transcutaneous oxygen partial pressures (tcPO2) of 20, 40 and 60 kPa. The magnitude of ventilation (ΔZv) and perfusion (ΔZQ)-related changes in cyclic impedance variations, were determined using an EIT prototype equipped with 32 electrodes around the thorax. Regional changes in ventral and dorsal right lung ventilation (V) and perfusion (Q) were estimated, and V/Q ratios calculated. RESULTS: There were no significant changes in ΔZv with increasing tcPO2 levels. ΔZQ in the dorsal lung increased with increasing tcPO2 (P = 0.01), whereas no such change was seen in the ventral lung. There was a simultaneous decrease in V/Q ratio in the dorsal region during hyperoxia (P = 0.04). Two subjects did not reach a tcPO2 of 60 kPa despite breathing 100% oxygen. CONCLUSION: These results indicate that breathing increased concentrations of oxygen induces pulmonary vasodilatation in the dorsal lung even at small increases in FiO2. Ventilation remains unchanged. Local mismatch of ventilation and perfusion occurs in young healthy men, and the change in ventilation/perfusion ratio can be determined non-invasively by EIT.

A glycyl radical site in the crystal structure of a class III ribonucleotide reductase.

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. Three classes have been identified, all using free-radical chemistry but based on different cofactors. Classes I and II have been shown to be evolutionarily related, whereas the origin of anaerobic class III has remained elusive. The structure of a class III enzyme suggests a common origin for the three classes but shows differences in the active site that can be understood on the basis of the radical-initiation system and source of reductive electrons, as well as a unique protein glycyl radical site. A possible evolutionary relationship between early deoxyribonucleotide metabolism and primary anaerobic metabolism is suggested.

Trisodium phosphonoformate, a new antiviral compound.

Trisodium phosphonoformate selectively inhibits cell-free DNA polymerase activity induced by herpesvirus. The new inhibitor has an antiviral effect on herpes simplex virus types 1 and 2, pseudorables virus, and infectious bovine rhinotracheitis virus in cell culture. It has a good therapeutic activity against cutaneous herpes simplex virus infection in guinea pigs.

Cardiac dysfunction after burns.

OBJECTIVES: Using transoesophageal echocardiography (TEE) we investigated the occurrence, and the association of possible abnormalities of motion of the regional wall of the heart (WMA) or diastolic dysfunction with raised troponin concentrations, or both during fluid resuscitation in patients with severe burns. PATIENTS AND METHODS: Ten consecutive adults (aged 36-89 years, two women) with burns exceeding 20% total burned body surface area who needed mechanical ventilation were studied. Their mean Baux index was 92.7, and they were resuscitated according to the Parkland formula. Thirty series of TEE examinations and simultaneous laboratory tests for myocyte damage were done 12, 24, and 36h after the burn. RESULTS: Half (n=5) the patients had varying grades of leakage of the marker that correlated with changeable WMA at 12, 24 and 36h after the burn (p< or =0.001, 0.044 and 0.02, respectively). No patient had WMA and normal concentrations of biomarkers or vice versa. The mitral deceleration time was short, but left ventricular filling velocity increased together with stroke volume. CONCLUSION: Acute myocardial damage recorded by both echocardiography and leakage of troponin was common, and there was a close correlation between them. This is true also when global systolic function is not deteriorated. The mitral flow Doppler pattern suggested restrictive left ventricular diastolic function.

The ribonucleotide reductase jigsaw puzzle: a large piece falls into place.

The three-dimensional structure of ribonucleotide reductase protein R1 from Escherichia coli reveals a novel 10-stranded alpha/beta barrel fold. A long loop penetrates the center cavity to assemble the active site cysteine triad.

Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding.

BACKGROUND: Ribonucleotide reductase (RNR) is an essential enzyme in DNA synthesis, catalyzing all de novo synthesis of deoxyribonucleotides. The enzyme comprises two dimers, termed R1 and R2, and contains the redox active cysteine residues, Cys462 and Cys225. The reduction of ribonucleotides to deoxyribonucleotides involves the transfer of free radicals. The pathway for the radical has previously been suggested from crystallographic results, and is supported by site-directed mutagenesis studies. Most RNRs are allosterically regulated through two different nucleotide-binding sites: one site controls general activity and the other controls substrate specificity. Our aim has been to crystallographically demonstrate substrate binding and to locate the two effector-binding sites. RESULTS: We report here the first crystal structure of RNR R1 in a reduced form. The structure shows that upon reduction of the redox active cysteines, the sulfur atom of Cys462 becomes deeply buried. The more accessible Cys225 moves to the former position of Cys462 making room for the substrate. In addition, the structures of R1 in complexes with effector, effector analog and effector plus substrate provide information about these binding sites. The substrate GDP binds in a cleft between two domains with its beta-phosphate bound to the N termini of two helices; the ribose forms hydrogen bonds to conserved residues. Binding of dTTP at the allosteric substrate specificity site stabilizes three loops close to the dimer interface and the active site, whereas the general allosteric binding site is positioned far from the active site. CONCLUSIONS: Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Structural basis for allosteric substrate specificity regulation in anaerobic ribonucleotide reductases.

BACKGROUND: The specificity of ribonucleotide reductases (RNRs) toward their four substrates is governed by the binding of deoxyribonucleoside triphosphates (dNTPs) to the allosteric specificity site. Similar patterns in the kinetics of allosteric regulation have been a strong argument for a common evolutionary origin of the three otherwise widely divergent RNR classes. Recent structural information settled the case for divergent evolution; however, the structural basis for transmission of the allosteric signal is currently poorly understood. A comparative study of the conformational effects of the binding of different effectors has not yet been possible; in addition, only one RNR class has been studied. RESULTS: Our presentation of the structures of a class III anaerobic RNR in complex with four dNTPs allows a full comparison of the protein conformations. Discrimination among the effectors is achieved by two side chains, Gln-114 and Glu-181, from separate monomers. Large conformational changes in the active site (loop 2), in particular Phe-194, are induced by effector binding. The conformational differences observed in the protein when the purine effectors are compared with the pyrimidine effectors are large, while the differences observed within the purine group itself are more subtle. CONCLUSIONS: The subtle differences in base size and hydrogen bonding pattern at the effector site are communicated to major conformational changes in the active site. We propose that the altered overlap of Phe-194 with the substrate base governs hydrogen bonding patterns with main and side chain hydrogen bonding groups in the active site. The relevance for evolution is discussed.

A small angle X-ray study of the elongation factor complex Tu . GDP from Escherichia coli.

The protein elongation factor complex Tu. GDP from Escherichia coli was investigated in the presence of 0.01 mM GDP using the small-angle X-ray method. The overall shape and the molecular parameters of the Tu . GDP complex were determined using a least-squares method where the experimental data were used directly without desmearing. The best fit to the experimental data was obtained assuming the molecule to be an ellipsoid of revolution with the semiaxes A = B = 4.08 nm, and C = 1.18nm. Determination of the molecular weight gave the result Mr = 46 000, which corresponds to a water content equal to 26% (by weight).

The solution shapes of IgG3 immunoglobulin and its Fch and Fc fragments. A small-angle X-ray scattering study.

A homogeneous IgG3 protein and its corresponding Fch and Fc fragments have been studied in solution using the small-angle X-ray scattering method. The Fch and Fc fragments were produced by short digestion of IgG3 with papain and trypsin. The results indicate that the overall shape of the IgG3 molecule in solution can best be described as an elliptical cylinder with a total length of 29 nm and with a cross-section having the semiaxes 3.8 and 0.9 nm. Thus, the overall shape of IgG3 is considerably different from the Y-shape normally adopted for the IgG1 molecule. The analysis of the data obtained for the Fch and Fc fragments also yields elliptical cylinders with almost the same dimensions of the cross-section but with shorter total lengths, 11.4 and 6.7 nm, respectively. The molecular weights of the IgG3 protein and the Fch and Fc fragments were determined to be 1.8 x 10(5), 0.61 x 10(5), and 0.50 x 10(5), respectively.

Temperature dependence of the kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into half-molecules. A minimum rate at 15 degrees C indicates hydrophobic interaction between the subunits.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 degrees C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native alpha 2-macroglobulin (delta CP is in the range 5 to 6 kJ mol-1 K-1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of alpha 2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation delta G has a maximum at about 32 degrees C, whereas the enthalpy of activation delta H and the entropy of activation delta S are zero at about 15 degrees C and 32 degrees C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.

Structure of RecA-DNA complexes studied by combination of linear dichroism and small-angle neutron scattering measurements on flow-oriented samples.

By combining anisotropy of small-angle neutron scattering (SANS) and optical anisotropy (linear dichroism, l.d.) on flow-oriented RecA-DNA complexes, the average DNA-base orientation has been determined in RecA complexes with double-stranded (ds) as well as single-stranded (ss) DNA. From the anisotropy of the two-dimensional SANS intensity representation, the second moment orientation function S is obtained. Knowledge of S is crucial for the interpretation of l.d. spectra in terms of orientation of the DNA bases and the aromatic amino acid residues. The DNA-base planes are essentially perpendicular to the fibre axis of the complex between RecA and dsDNA in the presence of cofactor ATP gamma S. A somewhat tilted base geometry is found for the RecA-ATP gamma S complexes with single-stranded poly(dT) and poly(d epsilon A). This behaviour contrasts the RecA-ssDNA complex formed without cofactor which displays a poor orientation of the bases. Well-ordered bases in the ssDNA-RecA complex is possibly reflecting the role of RecA in preparing a nucleotide strand for base-pairing in the search-for-homology process. While the central SANS intensity is essentially independent of the pitch of the helical complex, a secondary intensity maximum, which becomes focused upon flow orientation, is found to be a sensitive measure of the pitch. The pitch values for the complexes compare well with cryo-electron microscopy results but are slightly larger than those seen for uranyl-stained samples.

Structure of a RecA-DNA complex from linear dichroism and small-angle neutron-scattering in flow-oriented solution.

Small-angle neutron-scattering (SANS) and ultraviolet linear dichroism (l.d.) were measured on identical samples of a RecA-double-stranded (ds) DNA complex, including cofactor adenosine 5'-O-thiotriphosphate, which were aligned by flow in two equivalent Couette devices made of niobium and silica, transparent to neutrons and to ultraviolet light, respectively. The SANS anisotropy indicates a modest orientation of the RecA-dsDNA fiber with the helix axis parallel to the flow field. By correlation with the corresponding l.d. of the DNA at the same orientation conditions, it is inferred that the DNA bases have a local orientation that is approximately perpendicular to the helix axis. By comparison with the worse orientation in single-stranded DNA-RecA, this conclusion suggests that the dsDNA in its complex with RecA is not strand separated, and may be accommodated as an essentially unperturbed, straight double helix running along the RecA polymer fiber. The SANS anisotropy is also found to support the assignment of a subsidiary intensity maximum as originating from the pitch of a helical fiber.

Methyl-RNA: an evolutionary bridge between RNA and DNA?

Given the apparent limitation of double-stranded RNA (dsRNA) genomes to about 30 kb, together with the complexity of DNA synthesis, it appears difficult for a dsRNA genome to encode all the information required before the transition from an RNA to a DNA genome. Ribonucleotide reductase itself, which synthesizes deoxyribonucleotides from ribonucleotides, requires complex protein radical chemistry, and RNA world genomes may have reached their limits of coding capacity well before such complex enzymes had evolved. The transition from RNA to DNA thus appears to require intermediate steps, and we suggest that the naturally occurring 2'-O-methylated RNA, with chemical properties intermediate between RNA and DNA, is a suitable candidate.

New crystal forms of the small subunit of ribonucleotide reductase from Escherichia coli.

The small subunit of ribonucleotide reductase from Escherichia coli has been crystallized in two new crystal forms. The form most suitable for X-ray analysis belongs to the orthorhombic space group P2(1)2(1)2(1). It has the cell dimensions 74.3 A, 85.5 A, 115.7 A and diffracts to about 2.1 A resolution. The asymmetric unit most probably contains one dimer. Absorption spectra of single crystals confirm that the crystals contain a binuclear iron center. Crystals of the iron-depleted apoenzyme have also been obtained.

Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison.

The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.

The tyrosyl free radical in ribonucleotide reductase.

The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical.

Subaortic flow profiles in aortic valve disease: a two-dimensional color Doppler study.

With time-corrected color Doppler echocardiography, the aortic subvalvular spatial flow velocity profile was registered in two perpendicular planes in 10 patients with aortic valve disease and in 5 healthy control subjects. Patients with predominant aortic valve stenosis had a fairly flat profile, and the subvalvular diameter, obtained from left parasternal two-dimensional tissue imaging, provided a good estimate of the mean of the two transverse flow axes. This explains the accuracy in determination of stroke volume and aortic valve area that is reported in studies on patients with aortic valve stenosis when the continuity equation is used. However, the use of apical pulsed Doppler ultrasound registrations from the left ventricular outflow tract and parasternal two-dimensional echocardiography for flow area calculation may introduce large errors in calculated stroke volume in certain patients with aortic regurgitation and in normal subjects, because of a non-flat spatial velocity profile or an inaccurate estimate of flow area.

Two-dimensional color Doppler flow velocity profiles can be time corrected with an external ECG-delay device.

Although two-dimensional ultrasound color flow imaging is often considered to be a real-time technique, the acquisition time for two-dimensional color images may be up to 200 msec. Time correction is therefore necessary to obtain correct flow velocity profiles. We have developed a time-correction method in which a specially designed unit detects the QRS complex from the patient and creates a trig pulse that is delayed incrementally in relation to the QRS complex. This trig pulse controls the acquisition of the ultrasound images. A number of consecutively delayed images, with known incremental delay between the sweeps, can thus be stored in the memory of the echocardiograph and transferred digitally to a computer. The time-corrected flow velocity profile is obtained by interpolation of data from the time-delayed profiles. The system was evaluated in a Doppler string phantom test. With this technique it is possible to study time-corrected flow velocity profiles without the need to alter existing ultrasound Doppler equipment.

Problems in timing of respiration with the nasal thermistor technique.

When one analyzes transvalvular and venous flow velocity patterns, it is important to relate them to respiration. For this reason a nasal thermistor technique is often used, although it is known that this signal is delayed in relation to intrathoracic pressure changes. The magnitude and variation in delay have not been investigated previously and were, therefore, studied in a model experiment in 10 normal subjects, in 10 patients with obstructive, and in 10 patients with restrictive pulmonary disease. Esophageal pressure variations measured with an air-filled balloon served as a gold standard for intrathoracic pressure changes. During basal conditions there was, for both patient groups and normal subjects, a considerable delay of the thermistor signal. The average delay for all subjects was 370 msec with a wide variation (from 120 to 720 msec). At higher breathing frequencies the delay shortened to 310 msec (P < 0.01) but there was still a wide variation (ranging from 200 to 470 msec). Theoretic calculations show that the delay caused by the respiratory system accounts for only a minor portion of the total delay. Model experiments confirmed that the response characteristics of the thermistor probes limit the accuracy in timing of respiration. The total delay with the investigated thermistor technique is too long and variable to fulfil clinical demands.