Exploring the influence of iron doping on structural, morphological and optical properties of chemical bath deposited cadmium selenide thin films for optoelectronic applications.

This research investigates the structural, morphological, and optical properties of Cadmium Selenide (CdSe) thin films deposited via the Chemical Bath Deposition (CBD) Technique, focusing on the impact of Iron (Fe) doping. Using Cadmium Chloride (CdCl2) and Ferrous chloride (FeCl2) as precursor materials, the research investigates how Fe doping affects the structural and photoelectric characteristics of the films. Employing various characterization methods including X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Fourier-Transform Infrared Spectroscopy (FTIR), and UV-Vis NIR spectroscopy, the study provides a comprehensive analysis of the films. XRD analysis confirms the formation of a cubic structure with a predominant orientation along the (111) plane, consistent with XRD peaks. Additionally, XRD data reveals the degradation of thin films post-annealing. Crystalline size and strain are determined using the Debye-Scherrer and Wilson formulae, while lattice constant and Size-strain plots are derived from X-ray line broadening. The average crystallite size ranges from 12 to 21 nm. Optical band gaps are found to be 2.25 eV, 2.91 eV, 2.87 eV, and 2.85 eV for the samples. Interestingly, a decrease in crystal size with increasing doping concentration correlates with a reduction in bandgap. This investigation offers valuable insights into the fabrication and characterization of CdSe thin films, particularly highlighting the impact of Fe doping on their structural and optical properties. Overall, this study provides valuable insights into the fabrication and characterization of CdSe thin films, emphasizing the importance of precise doping control for tailoring material properties and advancing their applications in photovoltaic and optoelectronic devices.

Effect of stabilizer content in different solvents on the synthesis of ZnO nanoparticles using the chemical precipitation method.

Zinc Oxide (ZnO) nanoparticles (NPs) have been synthesized by a simple chemical precipitation method. The effect of monoethanolamine (MEA) content in different solvents on ZnO NPs synthesis and their structural properties has been investigated. The NPs synthesized by using isopropanol (IPA) with 15 ml MEA as a stabilizer under the most favorable conditions (deposition time, td = 120 min, temperature = 60 °C) showed good structural properties. Synthesized NPs exhibited beneficial structural properties after annealing. The hexagonal wurtzite crystal structure of ZnO NPs was verified by XRD. Different models were used to calculate structural parameters such as crystallite size, strain, stress, and energy density for all the reflection peaks of XRD corresponding to ZnO lying in the range 2θ = 15°-80°. The crystallite size of the ZnO nanoparticles was found to be 50-60 nm. FTIR and EDX confirmed the presence of ZnO NPs in the samples. SEM micrograph of all the samples revealed that the grain sizes decrease gradually with the increase of the amount of MEA. UV-Visible diffuse reflectance spectroscopy results provide evidence that the ZnO NPs possess broader absorption bands, together with high band gap energy. The ZnO NPs synthesized with IPA solvent have the highest transmittance and band gap energy of 3.3eV. According to DLS data, various content of MEA stabilizer in solvent affects the hydrodynamic size of ZnO NPs.

Cervical vertebrae maturation: a blinded trial study.

This blinded trial was conducted to analyze possible correlations between the cervical vertebrae maturation method (CVM) and the mineralization of mandibular teeth as described by Demirjian et al. (TMS). Panoramic and cephalometric radiographs of 500 orthodontic patients were analyzed by two blinded operators. TMS was utilized to analyze mineralization of second molar, second and first premolar and canine on the left side of the mandible; CVM stage was also evaluated. A blinded statistician performed statistical correlations and multiple regression analysis. Significant relations between CVM and TMS stages were identified for each tooth. Significant age differences resulted for CVM, second molar and second premolar (p<0.05). Significant correlations for second molar were observed between TMS D and CVM I-II, TMS G and CVM III, TMS H and CVM V-VI (p less than 0.01). Second molar stage G for both sexes indicates the ongoing of growth spurt. Stage G for boys and stage H for girls correlate significantly with the late part of PGS.

Illumina short-read and MinION long-read WGS to characterize the molecular epidemiology of an NDM-1 Serratia marcescens outbreak in Romania.

Background and Objectives: Serratia marcescens is an emerging nosocomial pathogen, and the carbapenemase blaNDM has been reported in several surveys in Romania. We aimed to investigate the molecular epidemiology of S. marcescens in two Romanian hospitals over 2010-15, including a neonatal NDM-1 S. marcescens outbreak. Methods: Isolates were sequenced using Illumina technology together with carbapenem-non-susceptible NDM-1-positive and NDM-1-negative Klebsiella pneumoniae and Enterobacter cloacae to provide genomic context. A subset was sequenced with MinION to fully resolve NDM-1 plasmid structures. Resistance genes, plasmid replicons and ISs were identified in silico for all isolates; an annotated phylogeny was reconstructed for S. marcescens. Fully resolved study NDM-1 plasmid sequences were compared with the most closely related publicly available NDM-1 plasmid reference. Results: 44/45 isolates were successfully sequenced (S. marcescens, n = 33; K. pneumoniae, n = 7; E. cloacae, n = 4); 10 with MinION. The S. marcescens phylogeny demonstrated several discrete clusters of NDM-1-positive and -negative isolates. All NDM-1-positive isolates across species harboured a pKOX_NDM1-like plasmid; more detailed comparisons of the plasmid structures demonstrated a number of differences, but highlighted the largely conserved plasmid backbones across species and hospital sites. Conclusions: The molecular epidemiology is most consistent with the importation of a pKOX_NDM1-like plasmid into Romania and its dissemination amongst K. pneumoniae/E. cloacae and subsequently S. marcescens across hospitals. The data suggested multiple acquisitions of this plasmid by S. marcescens in the two hospitals studied; transmission events within centres, including a large outbreak on the Targu Mures neonatal unit; and sharing of the pKOX_NDM1-like plasmid between species within outbreaks.

Assessment of Lung Ultrasound B-Lines in Dogs with Different Stages of Chronic Valvular Heart Disease.

BACKGROUND: In dogs with chronic valvular heart disease (CVHD), early recognition of pulmonary edema (PE) is of paramount importance. Recent studies in dogs showed that lung ultrasound examination (LUS) is a useful technique to diagnose cardiogenic PE. OBJECTIVES: To describe LUS features in dogs with different stages of CVHD, and to determine its diagnostic accuracy in detecting PE using thoracic radiography as the reference standard. ANIMALS: Sixty-three dogs with CVHD. METHODS: Prospective, multicenter, cross-sectional study. Each dog underwent physical examination, echocardiography, thoracic radiography, and LUS. The LUS findings were classified as absent, rare, numerous, or confluent B-lines. Sensitivity, specificity, and positive and negative predictive values of LUS B-lines to identify PE were calculated using thoracic radiography as the reference standard. RESULTS: Dogs in stage B1 had absent or rare B-lines in 14 of 15 cases (93.3%). Dogs in stage B2 had absent or rare B-lines in 16 of 18 cases (88.9%). All dogs in stage C, without radiographic signs of PE, had absent or rare B-lines. Dogs in stage C, with radiographic signs of PE, had numerous or confluent B-lines in 18 of 20 cases (90%). Lung ultrasound examination detected PE with a sensitivity of 90%, specificity of 93%, and with positive and negative predictive values of 85.7 and 95.2%, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Lung ultrasound examination showed good diagnostic accuracy to identify cardiogenic PE and might be helpful in staging dogs with CVHD. Lung ultrasound examination should be considered as a new, noninvasive diagnostic tool for clinicians managing CVHD in dogs.

Species distribution and susceptibility profile to fluconazole, voriconazole and MXP-4509 of 551 clinical yeast isolates from a Romanian multi-centre study.

This is the first multi-centre study regarding yeast infections in Romania. The aim was to determine the aetiological spectrum and susceptibility pattern to fluconazole, voriconazole and the novel compound MXP-4509. The 551 isolates were identified using routine laboratory methods, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and DNA sequence analysis. Susceptibility testing was performed using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) method and breakpoints. The yeasts originated from superficial infections (SUP, 51.5 %), bloodstream infections (BSI, 31.6 %) and deep-seated infections (DEEP, 16.9 %), from patients of all ages. Nine genera and 30 species were identified. The 20 Candida species accounted for 94.6 % of all isolates. C. albicans was the overall leading pathogen (50.5 %). Lodderomyces elongisporus is reported for the first time as a fungaemia cause in Europe. C. glabrata and Saccharomyces cerevisiae, as well as the non-Candida spp. and non-albicans Candida spp. groups, showed decreased fluconazole susceptibility (<75 %). The overall fluconazole resistance was 10.2 %. C. krusei accounted for 27 of the 56 fluconazole-resistant isolates. The overall voriconazole resistance was 2.5 % and was due mainly to C. glabrata and C. tropicalis isolates. Fluconazole resistance rates for the three categories of infection were similar to the overall value; voriconazole resistance rates differed: 4 % for BSI, 3.2 % for DEEP and 1.4 % for SUP. The antifungal activity of MXP-4509 was superior to voriconazole against C. glabrata and many fluconazole-resistant isolates. There was a large percentage of non-albicans Candida isolates. A large part of the high fluconazole resistance was not acquired but intrinsic, resulting from the high percentage of C. krusei.

The pathogenic L392V mutation of presenilin 1 decreases the affinity to glycogen synthase kinase-3 beta.

Determination of the effects of presenilin 1 (PSEN1) mutations, involved in autosomal dominant early-onset Alzheimer's disease (ADEOAD), on the interaction between PSEN1 and binding proteins is essential to determine which interactions are involved in Alzheimer's disease (AD) pathogenesis. The PSEN1 binding protein glycogen synthase kinase-3 beta (GSK-3 beta) has been considered as a key protein in AD pathogenesis since GSK-3 beta phosphorylates tau and hyperphosphorylated tau is a main component of neurofibrillary tangles associated to AD. We show here, using surface plasmonic resonance, that the pathogenic L392V mutation, identified in a large French ADEOAD pedigree including 39 affected members, leads to a decreased affinity to GSK-3 beta. We conclude therefore that the increase of affinity of PSEN1 to GSK-3 beta reported in previous studies is not a common effect of pathogenic mutations associated to ADEOAD.

Measles epidemic in Romania, 1996-1998: assessment of vaccine effectiveness by case-control and cohort studies.

A measles epidemic occurred in Romania with 32,915 cases and 21 deaths reported between November 1996 and June 1998, despite high vaccination coverage since the early 1980s. Most cases were unvaccinated children aged <2 years and vaccinated school-aged children. A case-control study among preschool children and a cohort study among primary-school children were conducted to estimate effectiveness of Romanian-produced measles vaccine, and to evaluate age at vaccination and waning immunity as risk factors for vaccine failure. Both studies indicated that measles vaccine was highly effective. One dose reduced the risk for measles by 89% (95% confidence interval (CI) 85, 91); two doses reduced the risk by 96% (95% CI 92, 98). Children vaccinated at <1 year of age were not at increased risk for measles compared with children vaccinated at > or =1 year. Waning immunity was not identified as a risk factor since vaccine effectiveness was similar for children vaccinated 6-8, 9-11, and 12-14 years in the past. Because specific groups were not at risk for vaccine failure, an immunization campaign that targets all school-aged children who lack two doses may be an effective strategy for preventing outbreaks. A mass campaign followed by increased first-dose coverage should provide the population immunity required to interrupt indigenous measles virus transmission in Romania.

PIP: Two studies examined the effectiveness of measles vaccines in Romania during the measles epidemic between 1996 and 1998. A case control study among preschool children and a cohort study among primary school children were conducted to estimate Romanian-produced vaccine effectiveness and to identify risk factors for measles among these age groups. Both studies found that measles vaccine was highly effective. Single-dose vaccine effectiveness was 89% and double-dose vaccine effectiveness was 96%. Univariate analysis of the case-control study indicated that being unvaccinated and being born of itinerant parents were significant risk factors for measles among preschool children. Children vaccinated at less than 1 year of age were not at increased risk for measles compared with children who receive the vaccine at 1 year or older. Because specific groups were not at risk for vaccine failure, an immunization campaign targeting all school-aged children who lacks two doses of measles vaccine may be an effective measure to prevent outbreaks in Romania.

The L392V mutation of presenilin 1 associated with autosomal dominant early-onset Alzheimer's disease alters the secondary structure of the hydrophilic loop.

Autosomal dominant early-onset Alzheimer's disease results mainly from mutations of the presenilin 1 (PSEN1) gene, which codes for an integral membrane protein of 467 amino acids. The hydrophilic loop (amino acids 263-407) of PSEN1, in which many pathogenic mutations have been localized, appears to be crucial for the protein function since it includes the binding domains to different PSEN1 partners. Using circular dichroism (CD) we analyzed the structural effects of the pathogenic L392V mutation and compared them with those of the E318G substitution. This study revealed that, the L392V mutation, in a phospholipidic medium which mimics the in vivo membrane environment, reduces the alpha helix content of the PSEN1 loop, whereas the E318G substitution, considered as a polymorphism, does not. These results suggest that the pathogenic effect of some PSEN1 mutations within the hydrophilic loop could be the alteration of the interaction to the different binding proteins through a disruption of the secondary structure.

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.

A survey on the vaccination practice for perinatal hepatitis B virus infection at a clinic in Okinawa, Japan.

The prevalence of hepatitis B (HB) virus carrier mothers and the HB immunization rate at a clinic in Okinawa prefecture were investigated. The prevalence of HB virus carrier mothers during 1981-1985, 1986-1994 and 1995-1997 were 3.5%, 2.3% and 1.1% respectively. According to the national prevention program, passive-active immunoprophylaxis with HB immune globulin (HBIG) and HB vaccine was carried out for 12 infants born to HB virus carrier mothers positive for HBe antigen (high risk infants) and for 24 infants born to carrier mothers negative for HBe antigen (low risk infants) since 1986. The immunization rate of high risk infants and low risk infants were 100.0% and 91.7%, respectively. A follow up system for the prevention program and assessment of vaccination schedule will be necessary to improve HB immunization rate of low risk infants in the future.

[Nontuberculous mycobacteria in the hospital environment]. / Micobacterii netuberculoase în mediul spitalicesc.

Mycobacteria other than tubercle (MOTT) are ubiquitous. Mycobacterial disease is most common at the extremes of age and at the patients with malignancy or HIV infection. In an attempt to establish effective measures to prevent nosocomial disease which has become increasingly important we investigated the presence of MOTT in hospital environment of oncological and pediatric units. Between 1994-1997, 21 strains of MOTT were isolated from 500 environmental specimens (4.2% positive samples). The sources of MOTT were tap water and dust. The most frequent species was Mycobacterium marinum.

Sequential and combined use of inactivated and oral poliovirus vaccines: Dolj District, Romania, 1992-1994.

To determine the feasibility of a vaccination strategy that would reduce the risk of vaccine-associated paralysis while retaining a barrier against the spread of wild poliovirus, a 2-year project was undertaken using enhanced-potency inactivated poliovirus vaccine (IPV) administered at 2 and 3 months of age followed by doses of both IPV and oral poliovirus vaccine (OPV) administered at 4 and 9 months of age. Vaccination coverage by 12 months of age with three or more doses of IPV and two doses of OPV among 16,566 infants eligible for vaccination was > 95% and > 80%, respectively. Among 51 children from whom blood samples were obtained 45 days after their third dose of IPV and first dose of OPV, 100% had serum neutralizing antibodies (reciprocal titer > or = 10) to all three poliovirus types. No cases of paralytic poliomyelitis due to either wild or vaccine-related strains were reported. The project demonstrated the feasibility, safety, and high immunogenicity of sequential use of IPV followed by OPV in Romania.

Conformational changes upon binding of a receptor loop to lipid structures: possible role in signal transduction.

The mas oncogene codes for a seven transmembrane helix protein. The amino acid sequence 253-266, from the third extracellular loop and beginning of helix 7, was synthesized either blocked or carrying an amino acid spin label at the N-terminus. Peptide binding to bilayers and micelles was monitored by ESR, fluorescence and circular dichroism. Binding induced tighter lipid packing, and caused an increase of peptide secondary structure. While binding to bilayers occurred only when peptide and phospholipid bore opposite charges, in micelles the interaction took place irrespective of charge. The results suggest that changes in lipid packing could modulate conformational changes in receptor loops related to the triggering of signal transduction.

Scorpion toxins as natural scaffolds for protein engineering.

A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins.

Solution structure of a green mamba toxin that activates muscarinic acetylcholine receptors, as studied by nuclear magnetic resonance and molecular modeling.

The three-dimensional solution structure of the MTX2 toxin (65 amino acids and 4 disulfides) from the green mamba venom (Dendroaspis angusticeps), a toxin that activates the pharmacological M1 muscarinic acetylcholine receptors, has been determined by nuclear magnetic resonance and molecular modeling. Seventeen structures were calculated from 810 distance and 68 dihedral angle restraints using DIANA and X-PLOR. The average rms deviation between the 17 refined structures and the energy-minimized average structure is 0.95 A for the backbone atoms. The overall folding of MTX2 consists of three loops stabilized by the four disulfides and forming a two- and a three-stranded beta-sheet. This structure appears to be very similar to that of other snake toxins, such as neurotoxins, fasciculins, and cardiotoxins, that also possess the same three-finger fold. For instance, the RMSd for the backbone atoms between MTX2 and the curaremimetic toxin alpha (from Naja nigricollis), the acetylcholinesterase inhibitor fasciculin 1 (from Dendroaspis angusticeps), and the cardiotoxic toxin gamma (from Naja nigricollis) are 1.86, 1.87, and 2.04 A, respectively. Local differences are observed between this toxin and the other structurally related toxins. Some of these differences could be relevant for the functional specificity of MTX2. In particular, this toxin presents a large twist at the tip of loop II due to a bulge (V31, T32; N35) that accommodates an inserted amino acid in the loop. This spatial arrangement brings the side chain of K34 in the beta-turn of the loop to be aligned with the beta-sheet. Hypotheses about a possible functional role of this lysine are described. Other characteristics in the side-chain distribution that could be related to the MTX2 function are presented.

Proton NMR studies of the structural and dynamical effect of chemical modification of a single aromatic side-chain in a snake cardiotoxin. Relation to the structure of the putative binding site and the cytolytic activity of the toxin.

This paper presents the comparative comprehensive analysis of NMR structural parameters (NOEs, scalar coupling, chemical shifts) of toxin gamma, a cardiotoxin isolated from the venom of Naja nigricollis, and three chemical derivatives, i.e. the 2-nitrophenylsulphonyl (NPS)-Trp11, 3-nitro-Tyr22 and 3-nitro-Tyr51 derivatives. In previous work, the chemical modifications of single side chains have suggested that these aromatic residues, in association with several lysine residues, contributed to the cytotoxicity of toxin gamma. Analysis of these results based on the refined solution structure of the toxin has resulted in the proposal of a conserved phospholipid binding site through which cardiotoxins are likely to interact with the membrane of target cells. The present work shows that modifications of either the tryptophan residue or the tyrosine residues, which are within or near the proposed binding site, have no influence on the three-dimensional structure of the protein. On the other hand, the proton exchange study of the backbone amides indicates that the structural core of the protein is destabilized in the three derivatives. This corresponds to a decrease of the overall stability of the protein as indicated by the comparative solvent denaturation study of the unmodified toxin gamma and the Trp11 derivative. More specifically, the dynamics of the three-stranded beta sheet, a part of the structural core, are highly perturbed by the chemical modifications. This sheet was previously proposed as a part of the phospholipid binding site of cardiotoxins. The dynamical perturbation of this site appears to be correlated with the decrease in toxicity of the chemical derivatives.

Binding of peptide fragments from a seven helix membrane receptor to lipid bilayers and to micelles.

Membrane proteins influence the organizational and motional properties of lipids, while the conformation and function of these proteins (receptors, channels, enzymes, pumps) are affected by the lipid environment. Model systems consisting of peptides and lipids can provide information at a molecular level about the interactions between proteins and lipids in biological membranes. We have synthesized peptides (residues 253-266 (EYWSTFGNLHHISL) from the seven-helix receptor expressed by the mas oncogene), having free or blocked N- and C-terminals. An analog was obtained by linking a spin-labeled amino acid to the N-terminal via a peptide bond. Several spectroscopic techniques were employed to study the interaction between the peptides and lipophilic systems (zwitterionic and negatively charged phospholipid bilayers, and negatively charged, positively charged, zwitterionic and nonionic micelles). Peptide conformational changes were monitored by circular dichroism (CD). The peptides acquired an increased secondary structure upon binding to the lipid systems. Additional evidence for peptide incorporation into micelles came from fluorescence measurements which indicated a blue shift of the tryptophan's emission wavelength, and from ESR spectra of the spin-labeled analog. While narrow lines were obtained in the aqueous phase, line broadening indicative of slower motion was observed in the presence of the lipophilic aggregates. The slow exchange between the two media allowed the evaluation of partition coefficients. The spectra in aqueous solution were also sensitive to conformational changes as a function of pH, allowing the determination of the N-terminal pK. ESR spectra of lipid spin probes incorporated into phospholipid bilayers indicated that the lipids became more immobilized upon binding of the peptides.

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.

Refined three-dimensional solution structure of a snake cardiotoxin: analysis of the side-chain organization suggests the existence of a possible phospholipid binding site.

The solution structure of toxin gamma (60 residues, 4 disulfides) from Naja nigricollis was determined by proton nmr and molecular modeling with DIANA and X-PLOR. The structures were calculated using 489 distance and 81 dihedral angle constraints. The average atomic rms deviation between the nine refined structures and the average structure is 0.118 nm for the backbone atoms. Toxin gamma has an overall folding consisting of three loops stabilized by the four disulfides and forming a two- and a three-stranded beta-sheet (loop I and loops II, III, respectively). The same type of folding has been observed for two homologous cardiotoxins. The very close similarity of the solution structure of toxin gamma and the crystal structure of toxin VII4 includes details of the topological arrangement of numerous side chains. Among these are the conserved residues K12, K18, K35, and Y22, known to be critical for the cytolytic activity of toxin gamma. A cluster of hydrophobic side chains organized around Y22 is found on one side of the three-stranded beta-sheet and is spatially close to a group of three lysines (K12, K18, K35). The side chains of these lysines form a cationic site that can accommodate the binding of a phosphate ion as found in the crystal structure of toxin VII4. The hydrophobic cluster constitutes a possible binding site for the hydrophobic moiety of phospholipids. Together with the complementary cationic site, this hydrophobic surface can form a conserved site by which cardiotoxins bind to membrane phospholipids.