Biomedical subjects

E Zak

Publications and source records attributed to E Zak.

15 recordsLinked to original sources

PubMed2001

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes.

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium Synechocystis sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.

Cell Membrane↗

PubMed2000

Dynamic interaction of plastocyanin with the cytochrome bf complex.

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.

Amino Acid Substitution↗

PubMed2000

The BtpA protein stabilizes the reaction center proteins of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 at low temperature.

Specific inhibition of photosystem I (PSI) was observed under low-temperature conditions in the cyanobacterium Synechocystis sp. strain PCC 6803. Growth at 20 degrees C caused inhibition of PSI activity and increased degradation of the PSI reaction center proteins PsaA and PsaB, while no significant changes were found in the level and activity of photosystem II (PSII). BtpA, a recently identified extrinsic thylakoid membrane protein, was found to be a necessary regulatory factor for stabilization of the PsaA and PsaB proteins under such low-temperature conditions. At normal growth temperature (30 degrees C), the BtpA protein was present in the cell, and its genetic deletion caused an increase in the degradation of the PSI reaction center proteins. However, growth of Synechocystis cells at 20 degrees C or shifting of cultures grown at 30 degrees C to 20 degrees C led to a rapid accumulation of the BtpA protein, presumably to stabilize the PSI complex, by lowering the rates of degradation of the PsaA and PsaB proteins. A btpA deletion mutant strain could not grow photoautotrophically at low temperature, and exhibited rapid degradation of the PSI complex after transfer of the cells from normal to low temperature.

Bacterial Proteins↗

PubMed1999

Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a large pigment-protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30-kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372-6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X-114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two-phase polymer partitioning technique to develop a novel method to isolate inside-out and right-side-out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.

Bacterial Proteins↗

PubMed1999

Progression and treatment of chronic adult periodontitis.

BACKGROUND: The periodontal status of 41 medically healthy adults with untreated chronic periodontitis was monitored before and after scaling and root planing (SRP). METHODS: During a 6-month pretreatment phase, clinical measurements, digital subtraction radiography (DSR) analysis of alveolar bone, and measurement of gingival crevicular fluid (GCF) prostaglandin E2 (PGE2) levels were undertaken. SRP was provided during a 1-month treatment phase. Clinical, radiographic, and biochemical analyses were repeated in a 6-month post-treatment healing period. RESULTS: Pretreatment: no clinically significant changes in mean plaque indices (PI), probing depths (PD), bleeding on probing (BOP), or relative clinical attachment levels (CAL) were detected (P>0.05). DSR revealed small but statistically significant bone height (0.04 mm) and mass (0.97 mg) loss (P<0.001). GCF PGE2 levels gradually increased from 38.8 ng/ml at month 1 to 79.4 ng/ml at month 6. Post-treatment: statistically and clinically significant reductions were observed in mean PI, BOP, and PD (P<0.05). A statistically significant reduction in CAL was noted (P<0.05). The trend towards progressive bone loss was halted and reversed, and a statistically significant decrease in GCF PGE2 concentrations was detected (P<0.001). Smokers, non-smokers, and ex-smokers did not differ significantly in PI, BOP, CAL, radiographic, or biochemical parameters at any time. Mean PD was significantly greater in current smokers than in non- and ex-smokers (P<0.005). PD reduced comparably in all 3 smoking subgroups following treatment (P<0.01). CONCLUSIONS: Conventional clinical measurements failed to identify disease progression over a 6-month period. Significant improvements were observed in clinical parameters after SRP, and a trend towards progressive bone loss was halted and reversed. Regular and frequent maintenance visits are important following treatment to maintain improvements in clinical parameters. Smokers had deeper probing depths than non- and ex-smokers, but pockets were reduced significantly and comparably in all 3 smoking subgroups following efficacious treatment.

Adult↗

PubMed1998

2D-isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803.

Aqueous polymer two-phase partitioning in combination with sucrose density centrifugation offered, for the first time, a 2D-separation method for the isolation of pure plasma and thylakoid membranes from the cyanobacterium Synechocystis 6803 without any cross-contaminations. The purity of the membrane fractions was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. As an initiation of a proteomics project, two prominent proteins, which were observed only in the plasma membrane (Slr1513, a hypothetical protein, and HofG, a general secretion pathway protein), or in the thylakoid membrane (PsaE, a photosystem I protein, and NdhH, a subunit of NADH dehydrogenase), were identified.

Bacterial Proteins↗

PubMed1998

Effects of ketorolac tromethamine mouthrinse (0.1%) on crevicular fluid prostaglandin E2 concentrations in untreated chronic periodontitis.

The effects of topical ketorolac tromethamine mouthrinse (0.1%) on gingival crevicular fluid (GCF) prostaglandin E2 (PGE2) concentrations were investigated in a 6-week, randomized, double-blind, placebo-controlled, parallel group, single center study of 42 patients with moderately advanced chronic adult periodontitis. Following screening, GCF was sampled from 6 sites per subject with filter paper strips and PGE2 levels measured using an enzyme immunoassay kit. Only those subjects with mouth median GCF PGE2 concentrations >30 ng/ml entered the rinsing phase. Eligible subjects were allocated placebo rinse in the first 2-week period (days 0 through 14), either ketorolac rinse (test group, n = 21) or placebo rinse (control group, n = 21) in the second 2-week period (days 14 through 28), and placebo rinse in the third 2-week period (days 28 through 42). Full mouth median GCF PGE2 concentrations were calculated for each subject at days 0, 14, 28, and 42, and group means were compared. From day 0 to day 14, no significant changes in GCF PGE2 concentrations were detected in either study group (P > 0.05). Utilizing mean GCF PGE2 concentrations at days 0 and 14 as covariates, no significant differences were observed in adjusted mean PGE2 levels at days 28 and 42 between the study groups (ANCOVA, P > 0.05). A statistically significant increase in GCF PGE2 levels was noted at days 28 and 42 in the placebo group (P < 0.01), but not in the ketorolac group (P > 0.05), when compared to baseline, however. GCF PGE2 levels were further studied in a subset of volunteers (n = 11) during a 12-hour period following first rinsing with mouthrinse (active or placebo) at day 14. GCF was sampled 0, 2, 4, 6, 8, and 12 hours post-rinsing. Mean PGE2 levels were higher in the placebo subgroup than in the ketorolac subgroup, and increased gradually over the 12-hour period in both subgroups. These data indicate that 1) 14 days of rinsing with 0.1% ketorolac mouthrinse controlled the elevation of GCF PGE2 observed in the placebo group but did not actually reduce GCF PGE2 concentrations and 2) changes in GCF PGE2 levels were not detectable in the 12-hour period following first rinsing with ketorolac.

Adult↗

PubMed1997

The achievement and maintenance of inter-examiner consistency in the assessment of plaque and gingivitis during a multicentre study based in general dental practices.

This study set out to demonstrate that it was possible to train general dental practitioners (gdps) to achieve and maintain high levels of inter-examiner consistency in the use of simple periodontal indices over a 12-month period. The gdps were trained by one trainer in the use of the plaque index (PII) and a modified version of the gingival index (mGI) which assessed gingival bleeding only. All the gdps underwent intensive training and employed a technique, when assessing inter-examiner consistency in the use of the PII, such that the 1st examiner did not disturb in situ plaque from 50% of the circumference of a tooth. The problem of variation due to repeat probing, when assessing inter-examiner consistency for mGI, was overcome by one examiner lightly probing gingival margins and both examiners scoring the results. The problems relating to the multicentre nature of the study included: distance between the centres, the need for strict adherence to the study protocol, consistency in the use of forms and instruments, in the application of periodontal indices, and of inclusion and exclusion criteria. Techniques for overcoming these problems included: the planning and application of a coherent study design, which employed simple indices, a detailed protocol, the recruitment of very well-motivated gdps of similar age and experience as examiners, the recruitment of an experienced trainer who trained the gdps thoroughly and monitored their performance throughout the study, and repeat visits to the practices involved to explain the nature of the study to all their staff members. Initially, the gdps achieved inter-examiner kappa scores of 0.78-0.85 (mean 0.81) for PII and of 0.73-0.94 (mean 0.87) for mGI when assessing 168 sites for each variable. During the following 12 months, individual kappa scores, assessed every 3 months at 42 sites, ranged from 0.51-0.90 for PII and from 0.73-1.00 for mGI. Mean kappa for PII scores achieved by the five gdps fell during the study from 0.81 to 0.76, whereas that for mGI rose from 0.87 to 0.92. It is concluded that it is possible to train gdps to achieve high levels of inter-examiner consistency in the use of PII and mGI and for these high levels to be maintained during a 12-month multicentre study.

Calibration↗

PubMed1997

The effects of a 0.12% chlorhexidine-digluconate-containing mouthrinse versus a placebo on plaque and gingival inflammation over a 3-month period. A multicentre study carried out in general dental practices.

Several previous studies have evaluated the effects of 0.12% chlorhexidine digluconate (ChD) mouthrinses on plaque and gingival inflammation. However, previously, none have been based in general dental practices. The aim of this study was to evaluate the potential to conduct controlled periodontal clinical trials in co-operation with general dental practitioners (gdps). The project took place in 5 general dental practices in the South of England. 121 healthy subjects (24 at 4 sites and 25 at the 5th), aged 18-65 years, mean 35 +/- 12) years participated in a double-blind, randomised study during which they received full mouth assessments for plaque and gingival bleeding at baseline, 6 and 12 weeks. 60 subjects were randomly assigned to use the 0.12% ChD mouthwash and 61 the placebo. The assessments were carried out by 5 gpds, who had previously achieved inter-examiner kappa scores of 0.78-0.85 (mean 0.81) for the plaque index (PII), and of 0.73-0.94 (mean 0.87) for a modified gingival index (mGI), and who maintained kappa scores of 0.51-0.90 for PII and of 0.73-1.00 for mGI during the 12 months required to complete the study. 98 subjects (48 ChD and 50 placebo) completed the study. Even though the baseline levels of plaque and gingivitis were low, by week 12, mean whole mouth plaque score of the ChD mouthwash users had fallen from 1.33 at baseline to 0.96 and was significantly lower (p < 0.001) than for the placebo users, 1.31 at baseline to 1.13. Whole-mouth gingival bleeding score fell from 0.56 to 0.42 in the ChD mouthwash group but was unchanged (0.54-0.55) in the placebo group. A subsidiary data analysis which considered the effects at sites indicated that within these overall differences, the ChD users experienced almost 2 x the reduction from plaque score 2 at baseline at proximal molar sites over a 12-week period (50.6% ChD versus 27.6% placebo). It was concluded that 0.12% ChD mouthwash reduced plaque accumulation by 28% and gingival inflammation by 25% over a 12-week period, that it is feasible for a group of gdps to maintain high levels of inter-examiner consistency in the use of PII and mGI, that it is also feasible to carry out such a multicentre study in general dental practice, and that the use of mean mouth scores per subject to analyse the effects of mouthrinses may well mask variations in response throughout the mouth.

Adolescent↗

PubMed1996

The plastocyanin binding domain of photosystem I.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.

Amino Acid Sequence↗

PubMed1984