Survey Study among Dentist in Bulgaria to Develop a Model for Risk Management in Dental Practice.

Background: Quality control and risk management in the field of dental services is an important part of improving patient safety as well as that of the dentists. The introduction of a risk management model would simplify and facilitate this process. Aim: The aim of the study is to gather information about the structurе and organization of work processes in Bulgarian dental practices, which will serve as a basis for building a risk management model. Material and Methods. A survey was conducted among randomly selected dental associations in Bulgaria-Plovdiv, Sofia, Varna, and Burgas through an anonymized questionnaire, containing 30 questions. The respondents meet the main criteria, namely, to be dentists and to practice in Bulgaria. The study includes demographic data, information on the attitude of Bulgarian dentists towards issues related to quality management, and safety and risk in respect to dental practice. The results have been processed and analyzed through descriptive and graphical analysis using the statistical program SPSS 20.0. Results: A total of 103 Bulgarian dentists took part in the study, out of which 25.24% ± 4.28% have acquired a specialty. Women are 52.43% ± 4.92%, and men -47.57% ± 4.92%. The largest is the relative share of the respondents in the age range of 25-35 years -63.10% ± 4.75% and with work experience of 6-15 years -52.43% ± 4.92%. Most of the respondents do not define in writing the main tasks and activities -52.43% ± 4.92%, and do not use checklists in their practice -54.73%. The majority of the respondents do not hold regular meetings with their teams -50.49% ± 4.93%, as well as they do not conduct surveys among their patients -68.93% ± 4.56%. The majority of the respondents -41.75% ± 4.86% are guided by their personal judgment in respect to whether the written information provided to patients is comprehensible and accessible. The majority of dentists -45.63% ± 4.91% take informed consent only for expensive procedures in written and oral form -53.40% ± 4.92%. Out of all the respondents, 75.73% ± 4.22% have not analyzed the risk of slipping in their practices for the last two years. Conclusion: There is a lack of written definition of the main tasks and processes, as well as no use of checklists in the practices of most of the interviewed dentists. Meetings with teams are held irregularly. There is a lack of surveys among patients, as well as no objective feedback from patients regarding the comprehensibility of the information materials provided. Informed consent is obtained from patients mainly in written and oral form and only for costly manipulations. In the practices of most of the interviewed dentists, there has been no assessment of the risk of slipping and falling for the last 2 years.

Occlusal Indicators Used in Dental Practice: A Survey Study.

INTRODUCTION: The function of the masticatory apparatus is complete when the dentition is intact with contact between the individual teeth and proper occlusion with the antagonists. For years, occlusal contacts have been studied to determine their exact location and describing various materials and methods for their registration such as paper foil, silk, and Shimstock foil. For years, occlusal contacts have been studied to determine their exact location and describe various materials and methods for their registration such as paper foil, silk, shim stock foil, the T-Scan system, and more recently the OccluSense system. The primary aim of the study was at evaluating which of the occlusal indicators is the most commonly used in practice, and the secondary aim was whether dentists are willing to use digital methods to examine occlusion. MATERIALS AND METHODS: The main primary information of the survey was collected by sending electronically anonymous questionnaires to 2014 dentists, randomly selected from all regions of the country. 228 questionnaires were filled in and returned. To achieve the goal of the study, the self-developed questionnaire was created and tested to survey the opinion about the use of occlusal indicators in dental practice. Each questionnaire contains questions about the sociodemographic and professional status of the people in the group and their opinion about the positives and negatives and the effectiveness of occlusal indicators. RESULTS: The obtained results confirm the statement that the most frequently used occlusal indicator in dental practice is the articulation paper. Articulation foil and silk are used less frequently than articulation paper. Of the listed quality indicators, Shimstock foil is rarely used in practice. Of the indicated quantitative indicators, the T-Scan system is more used than the OccluSense system. In the era of rapid technology development, the opinion and desire of dentists to increasingly want to introduce in their clinical practice quantitative methods are the digital diagnosis of occlusion. CONCLUSION: In any dental practice, if technically possible, digital methods would be used, giving more accurate and reliable data on the registered occlusal contacts.

Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product.

Chinese hamster ovary (CHO) cells are often used to produce therapeutic monoclonal antibodies (mAbs). CHO cells express many host cell proteins (HCPs) required for their growth. Interactions of HCPs with mAbs can sometimes result in co-purification of trace levels of 'hitchhiker' HCPs during the manufacturing process. Purified mAb-1 product produced in early stages of process optimization had high HCP levels. In addition, these lots formed delayed-onset particles containing mAb-1 and its heavy chain C-terminal fragments. Studies were performed to determine the cause of the observed particle formation and to optimize the purification for improved HCP clearance. Protease activity and inhibitor stability studies confirmed that an aspartyl protease was responsible for fragmentation of mAb-1 resulting in particle formation. An affinity resin was used to selectively capture aspartyl proteases from the mAb-1 product. Mass spectrometry identified the captured aspartyl protease as CHO cathepsin D. A wash step at high pH with salt and caprylate was implemented during the protein A affinity step to disrupt the HCP-mAb interactions and improve HCP clearance. The product at the end of purification using the optimized process had very low HCP levels, did not contain detectable protease activity, and did not form particles. Spiking of CHO cathepsin D back into mAb-1 product from the optimized process confirmed that it was the cause of the particle formation. This work demonstrated that process optimization focused on removal of HCPs was successful in eliminating particle formation in the final mAb-1 product.

Characterization of the initial level and migration of silicone oil lubricant in empty prefilled syringes for biologics using infrared spectroscopy.

Glass prefillable syringes are lubricated with silicone oil to ensure functionality and a consistent injection for the end user. If excessive silicone is applied, droplets could potentially result in aggregation of sensitive biopharmaceuticals or clouding of the solution. Therefore, monitoring and optimization of the applied silicone layer is critical for prefilled syringe development. The hydrophobic properties of silicone oil, the potential for assay interference, and the very small quantities applied to prefilled syringes present a challenge for the development of a suitable assay. In this work we present a rapid and simple Fourier transform infrared (FTIR) spectroscopy method for quantitation of total silicone levels applied to prefilled syringes. Level-dependent silicone oil migration occurred over time for empty prefilled syringes stored tip-up. However, migration from all prefilled syringes with between 0.25 and 0.8 mg of initial silicone oil resulted in a stable limiting minimum level of between 0.15 and 0.26 mg of silicone in the syringe reached after 1 to 4 years of empty tip-up storage. The results of the FTIR assay correlated well with non-destructive reflectometry characterization of the syringes. This assay can provide valuable data for selection of a robust initial silicone oil target and quality control of prefilled syringes intended for biopharmaceuticals. LAY ABSTRACT: Glass prefillable syringes are lubricated with silicone oil to ensure functionality and a consistent injection for the end user. If excessive silicone is applied, droplets could potentially result in aggregation of sensitive biopharmaceuticals or clouding of the solution. Therefore, monitoring and optimization of the applied silicone layer is critical for prefilled syringe development. The hydrophobic properties of silicone oil, the potential for assay interference, and the very small quantities applied to prefilled syringes present a challenge for the development of a suitable assay. In this work we present a rapid and simple Fourier transform infrared (FTIR) spectroscopy method for quantitation of total silicone levels applied to prefilled syringes. Level-dependent silicone oil migration occurred over time for empty prefilled syringes stored tip-up. However, migration from all prefilled syringes with between 0.25 and 0.8 mg of initial silicone oil resulted in a stable limiting minimum level of between 0.15 and 0.26 mg of silicone in the syringe reached after 1 to 4 years of empty tip-up storage. The results of the FTIR assay correlated well with non-destructive reflectometry characterization of the syringes. This assay can provide valuable data for selection of a robust initial silicone oil target and quality control of prefilled syringes intended for biopharmaceuticals.

Cross-linked silicone coating: a novel prefilled syringe technology that reduces subvisible particles and maintains compatibility with biologics.

Prefilled syringes (PFSs) offer improvements in the delivery of drugs to patients compared with traditional vial presentations and are becoming necessities in an increasingly competitive biologics market. However, the development of a product in a PFS must take into account potential incompatibilities between the drug and the components of the syringe. One such component is silicone oil, which has previously been suggested to promote protein aggregation, loss of soluble protein, and an increase in the particulate content of injectable formulations. This study evaluated the particulate content in a model buffer system (polysorbate 80/phosphate-buffered saline) after agitation in glass syringes with a novel cross-linked silicone coating. We also evaluated the compatibility of two monoclonal antibodies with these syringes. We report that syringes with this novel coating, compared with standard siliconized syringes, exhibited reduced particle content and enhanced integrity of the lubricant layer as determined by reflectometry, optical microscopy, and time-of-flight secondary ion mass spectrometry measurements, while maintaining the desired functional properties of the syringe and the antibodies' stability profiles as determined by high-performance size-exclusion chromatography. Enhanced integrity of the lubricant coating led to significantly fewer subvisible particles in the liquid formulations, particularly after agitation stresses introduced by shipping of the syringes.

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Protein particles: what we know and what we do not know.

All therapeutic protein products contain intrinsic particles formed by the aggregation of protein monomers. There is growing interest in understanding particles in biopharmaceutical products, fostered on one hand by significant advancements in particle analysis and on the other hand by concerns about potential impact of particles on product quality and safety. With currently available methods, particles in therapeutic proteins can be counted, sized, and characterized in a rudimentary way over a broad size range (from 10s of nanometers to 100 s of micrometers). Here, we review the known attributes of common protein particles, and then discuss the gaps in our current knowledge. The capabilities, limitations, and opportunities for improvement of common particle counting and characterization methods are listed. We conclude that further analytical progress is needed to better classify and characterize the diversity of particles encountered in therapeutic proteins, which may vary in the degree of protein unfolding, the inclusion of nonprotein nucleation centers, and aggregate morphology. Very little is known about the potential correlation between specific particle attributes and increased immunogenicity. In this environment of uncertainty, a deeper understanding about specific particle attributes and potentially increased immunogenicity is greatly needed and will likely be an area of future intensive research.

Effects of surfaces and leachables on the stability of biopharmaceuticals.

Therapeutic proteins are exposed to various potential contact surfaces, particles, and leachables during manufacturing, shipping, storage, and delivery. In this review, we present published examples of interfacial- or leachable-induced aggregation or particle formation, and discuss the mitigation strategies that were successfully utilized. Adsorption to interfaces or interactions with leachables and/or particles in some cases has been reported to cause protein aggregation or particle formation. Identification of the cause(s) of particle formation involving minute amounts of protein over extended periods of time can be challenging. Various formulation strategies such as addition of a nonionic surfactant (e.g., polysorbate) have been demonstrated to effectively mitigate adsorption-induced protein aggregation. However, not all stability problems associated with interfaces or leachables are best resolved by formulation optimization. Detectable leachables do not necessarily have any adverse impact on the protein but control of the leachable source is preferred when there is a concern. In other cases, preventing protein aggregation and particle formation may require manufacturing process and/or equipment changes, use of compatible materials at contact interfaces, and so on. This review summarizes approaches that have been used to minimize protein aggregation and particle formation during manufacturing and fill-finish operations, product storage and transportation, and delivery of protein therapeutics.

A study of pregnant women's knowledge of children's feeding practice as a risk factor for early childhood caries.

AIM: The aim of the present study was to assess the knowledge pregnant women have of infant and baby's feeding as a risk factor for early childhood caries. MATERIAL AND METHODS: The study included 200 pregnant women from Plovdiv and the region aged 18 to 41 with different educational backgrounds. A questionnaire was administered to all participants with question related to feeding babies and small children. The results were analysed using alternative analysis, non-parametric (chi2) test, Student t-test and graphic analysis. P < 0.05 was adopted as the level of significance of null hypothesis. RESULTS: The results show a low level of the knowledge pregnant women have of feeding as a potential risk factor for early childhood caries. A great percentage of the women with second and subsequent pregnancies gave wrong answers to at least one of the questions in the questionnaire. CONCLUSION: Based on the results of the study there is much reason to think that many pregnant women have inadequate knowledge of infant feeding. Introduction of educational programmes is essential with the purpose of raising the mothers' health care awareness in relation to children's dental health.

Solid state fluorescence of lyophilized proteins.

Fluorescence spectroscopy has been used to measure changes in the tertiary structure of proteins in the solution state. The sensitivity of fluorescence to the protein tryptophan environment has made it a useful tool for studying protein conformation and stability. Using fluorescence spectroscopy to probe structural alterations in lyophilized proteins has been limited due to technical challenges and overwhelming background light scattering. We have investigated the possibility of analyzing lyophilized proteins using the Cary-Eclipse spectrofluorometer by monitoring the fluorescence of the protein therapeutic after subjecting the lyophilized cake to heat-induced accelerated degradation. We have been able to obtain reproducible fluorescence spectra, detecting possible structural changes under these conditions. Fluorescence and circular dichroism spectroscopic analyses of the reconstituted proteins indicated that changes in fluorescence intensities observed in the solid state could be correlated to that in solution and to possible tertiary structural changes. Size exclusion chromatography analysis of protein Y subject to accelerated degradation showed a correlation between decreasing fluorescence intensity and increasing protein Y tetramer in solution, consistent with long-term stability. This suggests that solid state, intrinsic protein fluorescence measurements using the Cary-Eclipse holder may be feasible for long-term stability studies and formulation development.

Prevalence of dental fluorosis among 4- to 14-year-old children from the town of Dimitrovgrad (Bulgaria).

INTRODUCTION: There has been no study on the prevalence of dental fluorosis in Bulgaria of today where people have free access to some fluoride-containing products. AIM: The aim of the study was to investigate the prevalence of dental fluorosis among children 4 to 14 years old from the town of Dimitrovgrad, where due to unsatisfactory qualities of tap water people consume bottled water including such with fluoride levels higher than 1.5 mg/l. PATIENTS AND METHODS: The study included 1504 randomly selected children. We analysed subjects with dental fluorosis according to Dean's modified criteria. The following severity levels were defined: 0 - normal; 0.5 - suspicious; 1 - very mild; 2 - mild; 3 - moderate; 4 - severe. Data were analyzed separately for the different types of dentitions. RESULTS: Results showed that 54.52% of all children included in the study had dental fluorosis in different degrees. Primary teeth were affected by dental fluorosis less frequently than permanent teeth (P < 0.001). In mixed dentition cases 41.41% of the children had fluorosis of permanent teeth only, 1.64% had dental fluorosis of primary teeth only and 12.50% had both their primary and permanent teeth affected. The proportion of individuals with the lowest degree of severity - 0.5, was the greatest both for the primary and permanent teeth. Comparison with the proportions of children with more severe degrees of fluorosis revealed significant differences (P < 0.001). CONCLUSION: The results of the study showed excessive fluoride intake during tooth development and suggested a need for further research of risk factors.

Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX.

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.

Oxidation of methionine residues in the prion protein by hydrogen peroxide.

Reaction of H(2)O(2) with the recombinant SHa(29-231) prion protein resulted in rapid oxidation of multiple methionine residues. Susceptibility to oxidation of individual residues, assessed by mass spectrometry after digestion with CNBr and lysC, was in general a function of solvent exposure. Met 109 and Met 112, situated in the highly flexible amino terminus, and key residues of the toxic peptide PrP (106-126), showed the greatest susceptibility. Met 129, a residue located in a polymorphic position in human PrP and modulating risk of prion disease, was also easily oxidized, as was Met 134. The structural effect of H(2)O(2)-induced methionine oxidation on PrP was studied by CD spectroscopy. As opposed to copper catalyzed oxidation, which results in extensive aggregation of PrP, this reaction led only to a modest increase in beta-sheet structure. The high number of solvent exposed methionine residues in PrP suggests their possible role as protective endogenous antioxidants.

CARMIL is a bona fide capping protein interactant.

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the alpha- and beta-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is approximately 0.4 microm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, approximately 200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer <--> dimer equilibrium with an association constant of 1.0 x 10(6) m(-1). Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins ( approximately 1 and 2 microm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.

Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli.

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg(2+)) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20 degrees C, 5 mM pyruvate (with 2 mM Mg(2+)) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg(2+) is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 10(8) to < 5 x 10(5) or 3 x 10(7) M(-1), respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg(2+) also increases domain coupling and the dimerization constant approximately 42-fold. With 2 mM Mg(2+) at 15-25 degrees C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with K(A)' approximately 10(6) M(-1) (deltaG' = -8.05 +/- 0.05 kcal/mole and deltaH = +3.9 kcal/mole at 20 degrees C); deltaC(p) = -0.33 kcal K(-1) mole(-1). The binding of PEP to EI(H189A) is synergistic with that of Mg(2+). Thus, physiological concentrations of PEP and Mg(2+) increase, whereas pyruvate and Mg(2+) decrease the amount of dimeric, active, dephospho-enzyme I.

Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin.

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.

Prevalence of early childhood caries and risk factors in children from 1 to 3 years of age in Plovdiv, Bulgaria.

INTRODUCTION: Available data show that Early Childhood Caries (ECC) has a very wide range of prevalence (5% to 55%). Contemporary studies investigate the specific etiologic factors contributing to the appearance of ECC. As these questions are inadequately addressed in the stomatological literature in Bulgaria, we decided to investigate them in the present study. AIM: To determine the prevalence of ECC and the risk factors in children aged 12 to 47 months in Plovdiv. METHODS: The study is representative by design and is conducted in compliance with the requirements of World Health Organization. It includes 370 children 1 to 3 years of age, selected randomly. The dental caries was diagnosed by the visual-tactile method with a dental explorer and mirror at the cavitation level. A survey for determining the risk factors for ECC is carried out among the mothers of all affected children. RESULTS: The results of the study demonstrate high prevalence of ECC in the studied populations--20.82% in 1-year-old children, 40.0% in 2-year-old children and 56.15% in 3-year-old children. The analysis of the questionnaire data shows that the knowledge of mothers about the appropriate feeding of their children is insufficient. The use of baby's comforter with honey is not the only risk factor for developing caries. It is ascertained that frequent consumption of quickly soluble carbohydrates, as well as their prolonged contact with the tooth surface is highly significant risk factors too. CONCLUSIONS: The prevalence of ECC is high in the studied populations. The results of the questionnaire survey demonstrate the need for recommending adequate feeding practices of children till the age of 3 years to their mothers.

Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degrees C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [ approximately 2 x 10(6) (M monomer)(-1)] compared to those of dephospho-EI and EI(H189A) at 20 degrees C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl(2) [K(A)' > or = 10(8) M(-1) at 4 or 20 degrees C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(2+) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (T(m)) for unfolding the C-terminal domain by approximately 18 degrees C and that for the N-terminal domain by approximately 9 degrees C to T(max) congruent with 63 degrees C, giving a value of K(D)' congruent with 3 microM PEP at 45 degrees C. PEP alone also promotes the dimerization of EI(H189A) but only increases T(m) approximately 5 degrees C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K(D)' congruent with 0.2 mM for PEP dissociation in the absence of Mg(2+) at 45 degrees C. In contrast, the dimerization constant of phospho-EI at 20 degrees C is the same in the absence and presence of 5 mM PEP and 2 mM MgCl(2). Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(2+) are important determinants of both the conformational stability and dimerization of dephospho-EI.

Size Dependence of Protein-Induced Flocculation of Phosphatidylcholine Liposomes.

The adsorption of lysozyme and cytochrome C on phosphatidylcholine liposomes essentially changes the physical properties of the phospholipid membranes and under certain circumstances greatly affects the stability of the colloid dispersion by inducing bridging liposome flocculation. This study was designed to examine experimentally the influence of liposome size on two kinetic parameters of the flocculation, its rate constant and activation energy. As the liposome radius increased in the range 50-500 nm, the activation energy tended to decrease, resulting in an increased flocculation rate, except for the flocculation of 400-nm liposomes, which was greatly impeded. The pronounced influence of the liposome size on the flocculation rate constant was evident, since a well-defined minimum in the kinetic rate of flocculation of 400-nm liposomes was detected experimentally. The obtained nonlinear radius dependencies of the flocculation rates and activation energies are interpreted in terms of the bridging mechanism of the protein-induced liposome flocculation and the supplementary concept of the stability of thin liquid films formed between approaching protein-adsorbed liposomes. Copyright 2000 Academic Press.