[Biophysical mechanisms of dehydration of nasal cavity secretion in inflammatory diseases of the upper respiratory tract]. / Biofizicheskie mekhanizmy degidratatsii sekreta polosti nosa pri vospalitel'nykh zabolevaniiakh verkhnikh dykhatel'nykh putei.

The purpose of the study is an experimental-theoretical study of the mechanisms of structuring the secretion of the nasal cavity, in the process of its dehydration in inflammatory diseases of the upper respiratory tract. The work shows the general patterns of dehydration of natural biological fluids and their model solutions. Simulation of dehydration of the secret allowed us to identify the most informative parameters of changes in its composition in pathology and to develop criteria for diagnosing the inflammatory process of the VDP using the wedge-shaped dehydration method. The study clarified the mechanisms of dehydration of biological fluid, which expanded the possibilities of diagnosing diseases of the VDP.

[Single-stage open tympanoplasty in the form of tympanosclerosis with fixation of the stapes]. / Odnoétapnaia timpanoplastika pri otkrytoi forme timpanoskleroza s fiksatsiei stremeni.

We used several variants of one-stage tympanoplasty, including our own method, according to which, in the absence of an anvil and malleus after mobilisation of the stapes, ossiculoplasty with partial or complete ossicular prosthesis and myringoplasty simultaneously preventively created a support (neo-malleus) in the thickness of the neotympanic membrane. To assess the effectiveness of different variants of single-stage tympanoplasty, the long-term functional results were compared in terms of 6 to 12 months after the intervention with preoperative indicators. Standard methods of statistical estimation with calculation of descriptive statistics and methods of statistical hypothesis testing based on nonparametric Wilcoxon criterion for related samples were used. All the variants of one-stage tympanoplasty used in the fixation of the stapes are effective and, in general, make it possible to achieve an improvement in auditory function. In the present study, this result was achieved in 61 of 86 patients with tympanosclerosis (71% (65%; 74%) of 95% CI). The functional results of tympanoplasty according to the developed method do not differ from the results of traditional variants with the use of prostheses, but the implementation of this variant of tympanoplasty in the absence of an anvil and hammer allows for the fixation of the stapes during the reoperative stapedotomy using a prosthesis of the piston type without additional surgical stage of the formation of the neomaleus.

[The question about the topical antibiotic therapy of acute rhinosinusitis]. / K voprosu o topicheskoi antibakterial'noi terapii ostrykh rinosinusitov.

Presented the results of the clinical study of 30 patients with moderate rhinosinusitis (13 (43.3%) men, 17 (56.7%) women, age from 18 to 68 years). Among those patients, the inflammation of one paranasal sinus was observed in 7 (23.3%) cases, polysinusitis was observed in 23 (76. 7%) cases. All patients were randomized into 2 groups of 15 people. In both groups, patients received systemic antibiotic therapy, nasal irrigation therapy, and NSAIDs. In the control group, topical decongestants were used; in the experimental group the antimicrobial drug Polydexa with phenylephrine was used as a local therapy. The purpose of the study was to evaluate the clinical efficacy of Polydexa with phenylephrine in the complex treatment of moderate acute rhinosinusitis. The evaluation criteria were statistically significant comparison of clinical and laboratory parameters of both groups. Confirmed the anti-inflammatory, antimicrobial effects of the drug, made conclusions about the significant clinical efficacy, tolerability, positive effect on mucociliary clearance and safety of nasal spray Polydexa with phenylephrine.

[Skull base defects multilayer plasty in patients with spontaneous cerebrospinal fluid leak: our experience]. / Opyt primeneniia mnogosloinykh transplantatov v plastike defektov osnovaniia cherepa u bol'nykh so spontannoi nazal'noi likvoreei.

Spontaneous cerebrospinal fluid (CSF) leak is one of the types of non-traumatic CSF leaks in which the etiologic factor is unknown. Skull base defects transnasal endoscopic plasty is the initial method of surgical repair of spontaneous cerebrospinal fluid leaks. METHODS: Forty-five patients with spontaneous CSF leaks were managed using multilayer transplant technique. The basic choice criteria of endoscopic transnasal surgical approach and materials to reconstruction of skull base defects were the size of defect and its localization. In all cases pediculated flaps in combination with free graft were used. RESULTS: In 43 (96%) cases CSF leaks was successfully managed in primary surgery, which indicates high efficiency of the described surgical interventions.

[The intraoperative monitoring of the gas pressure in the middle ear].

The objective of the present work was to elucidate the patterns and mechanisms underlying the changes of gas pressure in the middle ear during the operation of stapedoplasty with a view to the application of the data obtained for objective recording of the stapedial reflex at the time of surgical intervention. Ten subjects with the unaffected hearing function were recruited for the study. Tympanometry was carried out at one-minute intervals starting from the onset of feeding nitrogen monooxide till the completion of septoplasty. It was shown that the surgical intervention under inhalation anesthesia is associated with periodic rises and drops in the gas pressure in the middle ear within a range from 500 dPA to 88 dPA with a period from 5.7 to 22 minutes. It is argued that these changes can be attributed to the periodic opening of the Eustachian tube when the gas pressure reaches a certain level due to continuous diffusion of nitrogen monooxide into the middle ear (the "preventive" release of pressure) followed by the passive closure of the auditory tube. The authors propose based on the results of the study recommendations on the performance of stapedial reflexometry during the surgical intervention with the use of impedancometry.

[Use of autotransplants and implants in ossiculoplasty].

The paper presents a comparative analysis of efficacy of ossiculoplasty (OP) using different transplants. A total of 202 operations were performed. Prostheses made of the fragments of the auditory ossicles, or a cortical layer of the temporal bone (n=81), of nail plate (n=56), titanium implants (n=65) were applied. A satisfactory result was achieved in 72-87% ossiculoplasties. Functional outcomes of OP did not vary significantly with type and material of the prosthesis. Main causes of poor OP results were fixation or displacement of the prosthesis with fibrous tissue; in case of titanium prostheses -- perforation of the tympanic membrane with prosthesis extrusion.

Nanospray 'taxation' and how to avoid it.

In mass spectrometric analysis with nanospray ionization, some analytes were found to appear in spectra with a delay of tens of minutes, while a few others could not be detected at all. The effect was found to be related to cation-exchange chromatography with negative charge on the glass surface, and with the most affected peptide or protein ions having strong localization of positive charge in blocks of two or more adjacent basic amino acid residues (e.g. melittin). The 'affinity' to the glass surface was studied with a peptide mixture and bovine serum albumin (BSA) tryptic digest solutions at sub-micromolar concentration. About 20% fewer tryptic peptides could be identified from spectra recorded with a glass nanospray capillary compared to those acquired with either conventional 1 microL/min electrospray or a quartz nanospray capillary. Protein identification studies are not likely to be seriously affected by this loss, but other protein applications, such as investigations of mutations or post-translational modifications, may suffer due to reduced sequence coverage. Ways to avoid losses of useful ions are discussed.

A high-performance matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometer with collisional cooling.

A high-performance orthogonal time-of-flight (TOF) mass spectrometer was developed specifically for use in combination with a matrix-assisted laser desorption/ionization (MALDI) source. The MALDI source features an ionization region containing a buffer gas with variable pressure. The source is interfaced to the TOF section via a collisional focusing ion guide. The pressure in the source influences the rate of cooling and allows control of ion fragmentation. The instrument provides uniform resolution up to 18,000 FWHM (full width at half maximum). Mass accuracy routinely achieved with a single-point internal recalibration is below 2 ppm for protein digest samples. The instrument is also capable of recording spectra of samples containing compounds with a broad range of masses while using one set of experimental conditions and without compromising resolution or mass accuracy.

Charge state separation for protein applications using a quadrupole time-of-flight mass spectrometer.

A novel method for separating ions according to their charge state using a quadrupole time-of-flight mass spectrometer is presented. The benefits of charge state separation are particularly apparent in protein identification applications at low femtomole concentration levels, where in conventional TOF MS spectra peptide ions are often lost in a sea of chemical noise. When doubly and triply charged tryptic peptide ions need to be filtered from singly charged background ions, the latter are suppressed by two to three orders of magnitude, while from 10-50% of multiply charged ions remain. The suppression of chemical noise reduces the need for chromatography and can make this experimental approach the electrospray equivalent of conventional MALDI peptide maps. If unambiguous identification cannot be achieved, MS/MS experiments are performed on the precursor ions identified through charge separation, while the previously described Q2-trapping duty cycle enhancement is tuned for approximately 1.4 of the precursor m/z to enhance intensities of ions with m/z values above that of the precursor. The resulting product ion spectra contain few fragments of impurities and provide quick and unambiguous identification through database search. The multiple charge separation technique requires minimal tuning and may become a useful tool for analysis of complex mixtures.

An introduction to quadrupole-time-of-flight mass spectrometry.

A brief introduction is presented to the basic principles and application of a quadrupole-time-of-flight (TOF) tandem mass spectrometer. The main features of reflecting TOF instruments with orthogonal injection of ions are discussed. Their operation and performance are compared with those of triple quadrupoles with electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometers. Examples and recommendations are provided for all major operational modes: mass spectrometry (MS) and tandem MS (MS/MS), precursor ion scans and studies of non-covalent complexes. Basic algorithms for liquid chromatography/MS/MS automation are discussed and illustrated by two applications.

Determination of the complete amino acid sequence for the coat protein of brome mosaic virus by time-of-flight mass spectrometry. Evidence for mutations associated with change of propagation host.

Time-of-flight mass spectrometry (TOFMS) has been applied to determine the complete coat protein amino acid sequences of a number of distinct brome mosaic virus (BMV) isolates. Ionization was carried out by both electrospray ionization and matrix-assisted laser desorption/ionization (MALDI). After determining overall coat protein masses, the proteins were digested with trypsin or Lys-C proteinases, and the digestion products were analyzed in a MALDI QqTOF mass spectrometer. The N terminus of the coat protein was found to be acetylated in each BMV isolate analyzed. In one isolate (BMV-Valverde), the amino acid sequence was identical to that predicted from the cDNA sequence of the "type" isolate, but deviations from the predicted amino acid sequence were observed for all the other isolates analyzed. When isolates were propagated in different host taxa, modified coat protein sequences were observed in some cases, along with the original sequence. Sequencing by TOFMS may therefore provide a basis for monitoring the effects of host passaging on a virus at the molecular level. Such TOFMS-based analyses assess the complete profiles of coat protein sequences actually present in infected tissues. They are therefore not subject to the selection biases inherent in deducing such sequences from reverse-transcribed viral RNA and cloning the resulting cDNA.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE).

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

[Effect of recombinant interleukin-1 beta on microbial flora of the middle ear in patients with chronic purulent otitis media]. / Vliianie rekombinantnogo interleikina-1 beta (betaleikina) na mikrobnuiu floru srednego ukha u bol'nykh khronicheskim gnoinym otitom.

Betaleukin was given to 60 patients with various forms of otitis media purulenta chronica (OMPC). Symptoms of the purulent exacerbation were relieved in 43.3% of the patients, the clinical course improved in 18.3%. No response was achieved in 40% of the treated patients. Betaleukin proved highly effective in management of exacerbations of uncomplicated OMPC though it has no direct antimicrobial activity.

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry.

The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.

SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.

Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei.

We have determined peptide sequences of three Trypanosoma brucei proteasome subunit proteins by mass spectrometry of tryptic digests of the proteins purified by two-dimensional (2-D) polyacrylamide gel electrophoresis. Three genes identified by the sequence of their cDNA encode the peptides identified in these three proteins. The three proteins predicted from the gene sequences have significant similarity to other known proteasome subunits and represent an alpha6 type subunit (TbPSA6), and two beta-type subunits belonging to the beta1-type (TbPSB1) and beta2 type (TbPSB2). The sequences of both beta-subunits predict formation of catalytically active subunits through proteolytic processing. The prediction is supported by the presence in each of the two beta-subunits of a tryptic peptide that has the correctly processed N-terminus that creates the threonine nucleophile of the mature protein. This peptide cannot be generated by trypsin because of the required cleavage of a glycine-threonine bond. It is thus likely that there are at least two catalytically active beta-subunits, TbPSB1 and TbPSB2, present in the mature 20S proteasome from T. brucei.

Peer Reviewed: Orthogonal-Injection TOFMS for Analyzing Biomolecules.

Orthogonal injection provides a high- efficiency interface for transferring ions from a continuous beam to a pulsed mode and makes it easier to obtain high resolution.

Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time-of-flight mass spectrometry.

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the trp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1:1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L- and D-tryptophan were also investigated. Two molecules of 5-methyl- or L-tryptophan were bound with high affinity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.

Electrospray mass spectrometry studies of non-heme iron-containing proteins.

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [subunit + Fe - 3H]+, and [subunit + 2Fe - 6 H]+. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer. Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da. Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.