Charge separation in bacterial photosynthetic reaction centers

The physical aspects of the primary charge separation process in bacterial photosynthesis are discussed. The donor-acceptor model of electron tranfer through proteins is used. The kinetics of the processes of the photosynthetic reaction centers are considered and their energetic scheme is constructed by means of the nonequilibrium density matrix method. It is shown that the theory is in good agreement with experiment if one takes into account the influence of vibrational sublevels of states which take part in transitions.     

In bacterial reaction centers rapid delivery of the second proton to QB can be achieved in the absence of L212Glu

In the reaction center (RC) of Rhodobacter capsulatus, residue L212Glu is a component of the pathway for proton transfer to the reduced secondary quinone, QB. We isolated phenotypic revertants of the photosynthetically incompetent (PS-) L212Glu-->Gln mutant; all of them retain the L212Glu-->Gln substitution and carry a second-site mutation: L227Leu-->Phe, L228Gly-->Asp, L231Arg-->Cys, or M231Arg-->Cys. We also characterized the L212Ala strain, which is a phenotypic revertant of the PS- L212Glu-L213Asp-->Ala-Ala mutant. The activities of the RCs of these strains--all of which lack L212Glu--were studied by flash-induced absorption spectroscopy. At pH 7.5, the rate of second electron transfer in the L212Q mutant is comparable to the wild-type rate. However, this mutant shows a marked decrease in the rate of cytochrome oxidation under strong continuous illumination and a very slow phase (0.66 s-1) of the proton transfer kinetics following the second flash, indicating that transfer of the second proton to QB is slowed more than 1000-fold. The levels of recovery of the functional capabilities in the revertant RCs vary widely; their rates of cytochrome oxidation were intermediate between those of the wild-type and the L212Q mutant. The kinetics of proton transfer following the second flash show a significant recovery in the L212Q + M231C and L212A RCs (330-540 s-1), but the L212Q + L227F RCs recover this function only partially. Compensation for the lack of L212Glu in revertant RCs is discussed in terms of (i) conformational changes that could allow water molecules to approach closer to QB and/or (ii) the increase in the negative electrostatic environment and the resultant rise in the free energy level of QB- that is induced by the mutations. The stoichiometries of H+/QB- proton uptake below pH 7.5 in the L212Q mutant, the L212Q + M231C revertant, and the wild-type strains are essentially equivalent, suggesting that L212Glu is protonated at neutral pH in wild-type RCs. This is also supported by the P+QB- charge recombination data. Comparison of H+/QB- proton uptake data with those obtained previously for the stoichiometries of H+/QA- proton uptake [Miksovska, J., Maróti, P., Tandori, J., Schiffer, M., Hanson, D. K., Sebban, P. (1996) Biochemistry 35, 15411-15417] suggests that L212Glu is the key to the electrostatic and perhaps structural interaction between the two quinone sites.     

Flash-induced changes in buffering capacity of reaction centers from photosynthetic bacteria reveal complex interaction between quinone pockets

The Abl-SH3 domain is implicated in negative regulation of the Abl kinase by mediating protein-protein interactions. High-affinity SH3 ligands could compete for these interactions and specifically activate the Abl kinase, providing control and a better understanding of the molecular interactions that underlie diseases where SH3 domains are involved. The p41 peptide (APSYSPPPPP) is a member of a group of peptide ligands designed to bind specifically the Abl-SH3 domain. It binds to Abl-SH3 with a Kd of 1.5 microM, whereas its affinity for the Fyn-SH3 domain is 273 microM. We have determined the crystal structure of the Abl-SH3 domain in complex with the high-affinity peptide ligand p41 at 1.6 A resolution. In the crystal structure, this peptide adopts a polyproline type II helix conformation through residue 5 to 10, and it binds in type I orientation to the Abl-SH3 domain. The tyrosine side-chain in position 4 of the peptide is hydrogen bonded to two residues in the RT-loop of the Abl-SH3 domain. The tight fit of this side-chain into the RT-loop pocket is enhanced by conformational adjustment of the main chain at position 5. The SH3 ligand peptides can be divided into two distinct parts. The N-terminal part binds to the SH3 domain in the region formed by the valley between the nSrc and RT-loops. It determines the specificity for different SH3 domains. The C-terminal part adopts a polyproline type II helix conformation. This binds to a well-conserved hydrophobic surface of the SH3 domain. Analysis of two "half"-peptides, corresponding to these ligand parts, shows that both are essential components for strong binding to the SH3 domains. The crystal structure of the Abl-SH3:p41 complex explains the high affinity and specificity of the p41 peptide towards the Abl-SH3 domain, and reveals principles that will be exploited for future design of small, high-affinity ligands to interfere efficiently with the in vivo regulation of Abl kinase activity.     

A new metal-binding site in photosynthetic bacterial reaction centers that modulates QA to QB electron transfer

Isolated reaction centers (RCs) from Rhodobacter sphaeroides were found to bind Zn(II) stoichiometrically and reversibly in addition to the 1 equiv of non-heme Fe(II). Metal and EPR analyses confirm that Zn(II) is ligated to a binding site that is distinct from the Fe site. When Zn(II) is bound to this site, electron transfer between the quinones QA and QB (QA-QB --> QAQB-) is slowed and the room-temperature kinetics become distributed across the microsecond to millisecond time domain. This effect of metal binding on the kinetics is similar to the more global effect of cooling RCs to 2 degreesC in the absence of Zn(II). This suggests that Zn(II) binding alters localized protein motions that are necessary for rapid QA-QB --> QAQB- electron transfer. Inspection of the RC crystal structure suggests a cluster of histidine ligands located beneath the QB binding pocket as a potential binding site.     

Pathway of proton transfer in bacterial reaction centers: role of aspartate-L213 in proton transfers associated with reduction of quinoneto dihydroquinone

The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that kAB(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced kAB(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pKa (pKa > or = 7) than Asp-L213 (pKa < or = 4), as indicated by the slower rate of charge recombination (D+QAQB(-)-->DQAQB) in the mutant RCs. The importance of the pKa and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried QB molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near QB.     

Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy

Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- charge-separated state. Fe-removed and Zn-substituted RCs from Rb. sphaeroides R-26 were prepared by refined procedures, and specific electron transfer rates (kQ) from the intermediate acceptor H- to the primary acceptor QA of (200 ps)-1 vs (3-6 ns)-1 were observed. Correlation of the transient EPR and optical results shows that the observed slow kQ rate in Fe-removed RCs is H-subunit-independent, and, in some cases, independent of Fe-site occupancy as Zn2+ substitution does not ensure retention of the native kQ. In addition, shifts in the optical spectrum of P865 and differences in the high-field region of the Q-band ESP spectrum for Fe-removed RCs with slow kQ indicate possible structural changes near P865. The experimental X-band and Q-band spin-polarized EPR spectra for deuterated Fe-removed RCs where kQ is at least 15-fold slower at room temperature than the (200 ps)-1 rate observed for native Fe-containing RCs have different relative amplitudes and small g-value shifts compared to the spectra of Zn-RCs which have a kQ unchanged from native RCs. These differences reflect the trends in polarization predicted from the sequential electron transfer polarization (SETP) model [Morris et al. (1995) J. Phys. Chem. 99, 3854-3866; Tang et al. (1996) Chem. Phys. Lett. 253, 293-298]. Thus, SETP modeling of these highly resolved ESP spectra obtained with well-characterized proteins will provide definitive information about any light-induced structural changes of P865, H, and QA that occur upon formation of the P865+QA- charge-separated state.     

Triplet energy transfer in bacterial photosynthetic reaction centres

[3-vinyl]-132-OH-bacteriochlorophyll a has been selectively exchanged against native bacteriochlorophyll a in the monomer binding sites at the A- and B-branch of the photosynthetic reaction centre from Rhodobacter sphaeroides. Transient absorption difference measurements were performed on these samples over a temperature range from 4.2 to 300 K with 20 ns time resolution. Specifically the decay of the primary donor triplet state, 3P870, as well as the rise and decay rates of the carotenoid triplet state, 3Car (spheroidene), were measured. The observed rates revealed a thermally activated carotenoid triplet formation corresponding to the decay of the primary donor triplet state. The activation energies for the triplet energy transfer process were 100(+/-10) cm-1 for reaction centers from wild-type Rhodobacter sphaeroides 2.4.1, with and without exchange of the monomeric bacteriochlorophyll on the electron transfer-active branch, BA. For reaction centers from Rhodobacter sphaeroides R26.1 with both monomers exchanged against [3-vinyl]-132-OH-bacteriochlorophyll a, and subsequent spheroidene reconstitution the activation energy was 460(+/-20) cm-1. These activation energies correspond to the energy difference between the triplet states of the accessory BChl monomer, BB, and the primary donor when native BChl a or [3-vinyl]-132-OH-BChl a is present in the BB binding site. In all samples the 3Car formation rates were bi-phasic over a large temperature range. A fast temperature-independent rate was observed on the wavelength of the carotenoid triplet-triplet absorption which dominated at very low temperatures. Additionally, a slower temperature-independent 3Car formation rate was observed at low temperatures which could be explained with the assumption of heterogeneity in the energy barrier (3BB) and/or the primary donor triplet state (3P870). A tunneling mechanism as proposed earlier by Kolaczkowski (PhD thesis, Brown University, 1989) is not only unnecessary but also incompatible with the available experimental data.     

Proton uptake by carboxylic acid groups upon photoreduction of the secondary quinone (QB) in bacterial reaction centers from Rhodobacter sphaeroides: FTIR studies on the effects of replacing Glu H173

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.     

Temperature dependence of the Qy resonance Raman spectra of bacteriochlorophylls, the primary electron donor, and bacteriopheophytins in the bacterial photosynthetic reaction center

Qy-excited resonance Raman spectra of the accessory bacteriochlorophylls (B), the bacteriopheophytins (H), and the primary electron donor (P) in the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides have been obtained at 95 and 278 K. Frequency and intensity differences are observed in the low-frequency region of the P vibrational spectrum when the sample is cooled from 278 to 95 K. The B and H spectra exhibit minimal changes of frequencies and relative intensities as a function of temperature. The mode patterns in the Raman spectra of B and H differ very little from Raman spectra of the chromophores in vitro. The Raman scattering cross sections of B and H are 6-7 times larger than those for analogous modes of P at 278 K. The cross sections of B and of H are 3-4 times larger at 95 K than at 278 K, while the cross sections of P are approximately constant with temperature. The temperature dependence of the Raman cross sections for B and H suggests that pure dephasing arising from coupling to low-frequency solvent/protein modes is important in the damping of their excited states. The weak Raman cross sections of the special pair suggest that the excited state of P is damped by very rapid (<30 fs) electronic relaxation processes. These resonance Raman spectra provide information for developing multimode vibronic models of the excited-state structure and dynamics of the chromophores in the RC.     

Solid state NMR studies of photoinduced polarization in photosynthetic reaction centers: mechanism and simulations

We simulate Photo-Chemically Induced Dynamic Nuclear Polarization in the 15N-solid-state NMR of 15N-labeled photosynthetic reaction centers using a Radical Pair Mechanism (RPM). According to the experimental data, the directly polarized nuclei include all eight nitrogens in the ground state of the bacteriochlorophyll special pair (P), and N-II in the bacteriopheophytin acceptor (H) [M.G. Zysmilich, A.E. McDermott, J. Am. Chem. Soc., 116 (1994) 8362-8363.] [M.G. Zysmilich, A. McDermott, J. Am. Chem. Soc., 118 (1996) 5867-5873.] [M.G. Zysmilich, A. McDermott, Proc. Natl. Acad. Sci. U.S.A., 93 (1996) 6857-6860.]; other signals are polarized in nonspecifically labeled samples, but the polarization apparently results from magnetization exchange with neighboring polarized nitrogens, and these are not treated in this work. Two quantitative models for the polarization associated with the RPM are presented and are used to test the validity of the proposal that this mechanism is cooperative in the reaction centers. The kinetic models can treat the steady state polarizations as well as the approach to steady state, and in principle could be expanded to include anisotropic effects, or pulse-probe experiments. Several features of the detailed simulations of the steady-state amplitudes and the kinetics of the approach to steady-state are compared with our data, including the signs and approximate absolute magnitudes of the polarization on the nitrogen nuclei in P and H(L), and the changes in the relative amplitudes with the change in the lifetime of the molecular triplet, photoaccumulation time, nuclear relaxation rate and illumination intensity. The simulations demonstrate that the polarization intensities are in qualitative agreement with those predicted for the RPM, including the curious observation of strong polariza-tion on the pheophytin acceptor for certain experimental conditions. However, this agreement requires efficient relaxation of the nitrogens on H(L) by 3P, due to a fortuitous low nanosecond value for the spin-lattice relaxation for the electrons in the molecular triplet of the donor, T1e of 3P. Whether this fortuitous match is valid is unproven.     

Low-temperature femtosecond-resolution transient absorption spectroscopy of large-scale symmetry mutants of bacterial reaction centers

Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.     

A new pathway for transmembrane electron transfer in photosynthetic reaction centers of Rhodobacter sphaeroides not involving the excited special pair

It is generally accepted that electron transfer in bacterial photosynthesis is driven by the first singlet excited state of a special pair of bacteriochlorophylls (P*). We have examined the first steps of electron transfer in a mutant of the Rhodobacter sphaeroides reaction center in which charge separation from P* is dramatically slowed down. The results provide for the first time clear evidence that excitation of the monomeric bacteriochlorophyll in the active branch of the reaction center (B(A)) drives ultrafast transmembrane electron transfer without the involvement of P*, demonstrating a new and efficient mechanism for solar energy transduction in photosynthesis. The most abundant charge-separated intermediate state probably is P+B(A)-, which is formed within 200 fs from B(A)* and decays with a lifetime of 6.5 ps into P+H(A)-. We also see evidence for the involvement of a B(A)+H(A)- state in the alternative pathway.     

Using action research to facilitate organisational change in mental health service delivery

OBJECTIVE: To evaluate human immunodeficiency virus (HIV)-1 RNA burden in paired plasma and cervicovaginal lavage specimens and to assess the relation of plasma HIV-1 RNA level, CD4 cell count, and antiretroviral therapy with cervicovaginal HIV-1 viral load. METHODS: Paired blood and cervicovaginal lavage specimens were collected from 72 HIV-infected women. Quantitation of HIV-1 RNA from plasma and cervicovaginal lavage specimens was performed by using the nucleic acid sequence-based amplification assay. Analyses examined relations between cervicovaginal HIV-1 RNA and plasma HIV-1 RNA level, CD4 count, and antiretroviral therapy. RESULTS: Plasma HIV-1 RNA was detectable in 61 of 72 women (85%), with copy numbers ranging from 330 to 1,600,000 copies/mL. Twenty-eight of 72 (39%) had detectable HIV-1 RNA in cervicovaginal lavage specimens, ranging from 320 to 440,000 copies/mL. The cervicovaginal lavage HIV-1 RNA level was detectable in 9%, 29%, 52%, and 53% of the women with plasma HIV-1 RNA of less than 400, 400-9999, 10,000-100,000, and more than 100,000 copies, respectively (P = .043). Among women with CD4 counts of less than 200, 200-500, and greater than 500/mm3, cervicovaginal lavage HIV-1 RNA was detected in 67%, 32%, and 25% of subjects, respectively (P = .018). Among women receiving antiretroviral therapy, cervicovaginal lavage revealed HIV-1 RNA in 67%, 31%, and 25% with CD4 cell counts of less than 200, 200-500, and more than 500/mm3, respectively (P = .042). CONCLUSION: The presence of HIV-1 RNA in cervicovaginal lavage correlates significantly with the level of HIV-1 RNA in plasma and negatively with CD4 cell count.     

Low frequency vibrational modes in proteins: changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm-1) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from Rhodobacter sphaeroides were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.     

Characterization of bacterial reaction centers having mutations of aromatic residues in the binding site of the bacteriopheophytin intermediary electron carrier

We report the initial characterization of a series of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter capsulatus having single or double mutations of phenylalanines 97 and 121 on the L polypeptide. Substitution of these aromatic amino acids, which may interact with the photoactive bacteriopheophytin associated with the L polypeptide (BPhL), was carried out to examine their possible roles in electron transfer, charge stabilization, and/or BPhL binding. In some mutant RCs, the wild-type pigment content is obtained while in certain others a bacteriochlorophyll (BChL) replaces BPhL. The mutant RCs with wild-type pigment content are found to have overall photochemistry effectively identical to that of wild-type RCs. This indicates aromatic residues at L97 and L121 are not critical factors in the charge separation process, although an approximate 2-fold increase in the rate of electron transfer from BPhL- to QA is observed in two mutants where residue L121 is leucine. In two double mutants where L121 is histidine and L97 is either valine or cysteine, BPhL is replaced with a BChl (denoted beta). This pigment content is surprising since in the native RC structure amino acid L121 is not in optimum geometry for coordination to the Mg in the center of the pigment macrocycle. Charge separation takes place in the beta-containing mutants with an approximately 70% yield of P+QA- at 285 K compared to approximately 100% for wild-type. The photochemistry of these new beta-type RCs is very similar to that reported previously for the beta RC from Rhodobacter sphaeroides wherein the same pigment change was induced by a mutation in the M polypeptide.     

Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Br?nsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Br?nsted plots were nonlinear. Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.     

Asthma among secondary schoolchildren in relation to the school environment

BACKGROUND: Poor indoor air quality has been suggested to be related to the increase in the prevalence of asthma that has occurred in the western world, especially among children and young persons. Apart from the home, school is the most important indoor environment for children. OBJECTIVES: The aims were to study the prevalence of current asthma among secondary pupils and its relationship to the school environment, but also to personal factors and domestic exposures. METHODS: Data on asthmatic symptoms, other health aspects, and domestic exposures were gathered using a questionnaire which was sent to 762 pupils in the seventh form (13-14 years old) in 11 randomly chosen schools in the county of Uppsala in Sweden. Pupils answering 'yes' to having had asthma diagnosed by a physician, and having had recent asthma attacks, or who used asthma medication were defined as having current asthma. Data on exposures at school were gathered by measurements in 28 classrooms. The relationship between asthma and exposures was analysed by multiple logistic regression. RESULTS: The questionnaire was completed by 627 (82%). Current asthma was found among 40 pupils (6.4%). Current asthma was more common in those who had an atopic disposition, or food allergy, or who had attended a day care centre for several years. Controlling for these factors, current asthma was related to several factors in the school environment. There were more pupils with current asthma in schools that were larger, had more open shelves, lower room temperature, higher relative air humidity, higher concentrations of formaldehyde or other volatile organic compounds, viable moulds or bacteria or more cat allergen in the settled dust. CONCLUSIONS: Although the pupils attended school for a minor part of their time, our study indicates that the quality of the school environment is of importance and may affect asthmatic symptoms.     

Mixed lipid-protein films of bacterial photosynthetic reaction centres. II. Mixed multilayers on solid supports

The cytokine interleukin 8 (IL-8) has been shown to be a potent mediator of leukocyte recruitment and neovascularization in inflammatory and neoplastic diseases. In this study we hypothesize that IL-8 produced in the nasal polyp microenvironment is responsible for the leukocyte recruitment seen in nasal polyposis. To test this hypothesis we evaluated nasal polyps for distribution and content of IL-8 antigen with immunohistochemical techniques and radioimmunoassay to determine tissue levels of IL-8. The immunohistochemical results demonstrated that IL-8 antigen staining occurred predominantly within inflammatory cells and epithelium. IL-8 was detected in all nasal polyp tissue homogenates (a mean value of 1767 +/- 1633 pg/mg total protein (TP) with a range of 134 to 3668 pg/mg TP vs control specimens with a mean value of 77 pg/mg TP with a range of 0.09 to 255 pg/mg TP). These data demonstrate the presence and distribution and levels of IL-8 antigen in nasal polyps in vivo, supporting our hypothesis that local production of IL-8 could be an important factor in the sustained recruitment of leukocytes in nasal polyposis. Thus IL-8 likely plays a significant role in the pathogenesis of this disease process and therefore is a potential target for therapeutic intervention.