[Digital technologies in the assessment of neurological status]. / Tsifrovye tekhnologii v otsenke nevrologicheskogo statusa.

Digital technologies (DT) can be used at all stages of the neurologist's work with the patient. The medical professional can obtain online information on the patient's complaints and history. DT may help to assess cognitive functions, muscular power, details of the movements including gate. The methods of the assessment of sensory functions are being currently developed. The methods of the assessment of the olfaction, vision, oculomotor function, pupillary reactions, mimic muscles, hearing and balance are also developed, however the methods of assessment of the function of trigeminal nerve, movement of the head, neck and tongue using DT are not available. The assessment of the reflexes using DT is not developed yet. The use of DT is possible in telemedicine, in long-term monitoring of the neurological status of the patient, as well as in the clinical exam in order to obtain additional, more detailed data.

[Transcranial magnet therapy in comprehensive therapy of migraine]. / Primenenie transkranial'noi magnitoterapii v kompleksnom lechenii migreni.

Migraine is a primary form of headache characterized by throbbing unilateral attacks, increased sensitivity to light and sound, accompanied by nausea and/or vomiting, lasting from 4 hours to 3 days. Developing and implementing new methods of pain relief is an urgent task of modern medicine. One of the safest and most commonly recommended methods is transcranial magnet therapy (TMT). OBJECTIVE: To evaluate the effect of TMT on improving the effectiveness of comprehensive therapy in patients with migraine. MATERIAL AND METHODS: A blind, randomized, placebo-controlled clinical trial was conducted, including 50 patients with migraine divided into three groups (the main group received TMT; the comparison group received low-frequency magnet therapy with a magnetic pulse duration of 250 µs; the control group received treatment with placebo device). The objectives were to study the TMT effect on reduction of the frequency and intensity of headache attacks, the severity of associated symptoms, reduction of medications use, including analgesics, in patients with migraine, as well as to evaluate the statistical difference in the effectiveness of magnetic therapy with different magnetic pulse ratio on all of the above parameters in study patients. RESULTS: According to the study data, a positive effect of TMT on the patients' condition was observed. After treatment, 76.9% of the patients in the main group had a reduction of headache intensity compared to the control group (35%) and a 47.8% decrease in analgesics use. In the main group, a reduction of nausea (73.3% of patients), acousticophobia (77.8% of patients), and photophobia (81.8% of patients) was noted. The HADS scale showed a 44.3% reduction in anxiety and depression in the main group. CONCLUSION: The clinical efficacy of TMT in patients with migraine has been demonstrated. In clinical practice, it is reasonable to use TMT to improve the treatment effectiveness in patients with migraine.

Co-morbidity in older patients with COPD--its impact on health service utilisation and quality of life, a community study.

BACKGROUND: co-morbidity has been shown to be an important consideration in COPD with an estimated prevalence of 84%. In the Netherlands, a weak association between health-related quality of life and lung function has been found, with a closer link to co-morbidity. OBJECTIVE: to determine the influence of co-morbidity on quality of life and health service utilisation in older patients with COPD in the community. DESIGN: observational cohort study. SETTING: general practice in the North East of England that has a list size of 8300. PARTICIPANTS: 27 patients aged 70 years or above on the practice COPD register. MEASUREMENTS: data on age and sex, spirometry to confirm the diagnosis of COPD, questionnaires to assess quality of life, activities of daily living (ADLs) and co-morbidity. Health service utilisation was recorded by the number of primary and secondary care attendances in the previous year. RESULTS: 10 had mild, 12 had moderate, and 5 had severe disease. Mean age was 76 years. Quality of life (QOL), co-morbidity and health service utilisation measurements were not significantly different between COPD severity groups. There was a significant positive correlation between increasing co-morbidity and poor QOL (r = 0.45, P < 0.05), and significant negative correlation between co-morbidity and ADL scores (scored inversely), r = -0.54, P < 0.05. Significant negative correlation was found between co-morbidity and primary care attendances (r = -0.41, P < 0.05) and significant positive correlation between worsening QOL and secondary care attendances (r = 0.46, P < 0.05). CONCLUSIONS: co-morbidity has an important part to play in COPD assessment, more accurately reflecting QOL in our population. Health service utilisation did not correlate to forced expiratory volume (FEV1)-defined COPD severity.

Beta blockers in older persons with heart failure: tolerability and impact on quality of life.

OBJECTIVE: To determine tolerability and symptom changes associated with the introduction of bisoprolol treatment in older patients with heart failure. DESIGN: Prospective observational cohort study. SETTING: Geriatric medicine outpatient department of a university hospital. PATIENTS: 51 patients (mean age 78 years, range 70-89 years) with stable symptomatic heart failure caused by left ventricular systolic dysfunction. INTERVENTIONS: Bisoprolol tablets, 1.25-10.0 mg. MAIN OUTCOME MEASURES: Tolerability; changes in symptoms and exercise tolerance. RESULTS: 69% of patients tolerated bisoprolol. Mean tolerated dose was 7.6 mg. There was no change in symptoms or exercise capacity in those who tolerated bisoprolol. Perceived health status and symptoms of anxiety and depression improved during the titration period. CONCLUSIONS: The rate of withdrawal from bisoprolol treatment in older patients with congestive heart failure was twice that previously reported in younger patients. The mean tolerated dose was similar to that found in trials reporting clinical efficacy. There was no evidence of a negative impact on symptoms or exercise capacity in patients who tolerated bisoprolol.

Bordetella pertussis adenylate cyclase toxin: a versatile screening tool.

The calmodulin-activated adenylate cyclase (AC) toxin is an essential virulence factor of Bordetella pertussis, the causative agent of whooping cough. This toxin has been exploited to devise screening techniques for investigating diverse biological processes. This mini-review describes several such applications. First, AC has been utilized as a selective reporter for protein translocation from bacteria to eukaryotic cells, in particular to study protein targeting by type III secretion machinery. More recently, AC has been used as a signal transducer in Escherichia coli to elaborate genetic screens for protein-protein interactions ("bacterial two-hybrid system") or site-specific proteolytic activities.

Protein-protein interaction between Bacillus stearothermophilus tyrosyl-tRNA synthetase subdomains revealed by a bacterial two-hybrid system.

We have recently developed a bacterial two-hybrid system (BACTH), based on functional complementation between two fragments of the catalytic domain of Bordetella pertussis adenylate cyclase (AC), that allows an easy in vivo screening and selection of functional interactions between two proteins in Escherichia coli. In this work, we have further explored the potentialities of the BACTH system to study protein-protein interactions, using as a model, the interactions between various subdomains of the dimeric tyrosyl-tRNA synthetase (TyrRS) of Bacillus stearothermophilus. Using the BACTH system we confirmed the known interactions of the alpha/beta domains and those between the alpha/beta domain and the alpha domain that could be anticipated from the three-dimensional structure of TyrRS. Interestingly, the BACTH system revealed the unexpected interaction between the TyrRS alpha domains which is presumably mediated by a pseudo-leucine zipper motif. This study illustrates the interest of the bacterial two-hybrid system to delineate interacting domains of proteins and shows that it can reveal interactions that occur in vivo and that were not anticipated from the three-dimensional structure of the protein of interest.

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8+ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.

Bordetella pertussis adenylate cyclase toxin as a tool to analyze molecular interactions in a bacterial two-hybrid system.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin (CyaA) that is able to enter into eukaryotic cells. We took advantage of the modular structure of the catalytic domain of CyaA to design a genetic system that can detect protein-protein interactions in Escherichia coli. This bacterial two-hybrid system is based on the functional complementation between two complementary fragments, T25 and T18, of the catalytic domain of CyaA, in an E. coli cya strain. This bacterial two-hybrid system could find applications in the studies of structure/function relationships of proteins, in functional analysis of genomes, in high-throughput screening of interacting ligands and in design of new therapeutic agents.

Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli.

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya(-) phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.

Genetic systems for analyzing protein-protein interactions in bacteria.

Analysis of protein-protein interactions has been revolutionized by the yeast two-hybrid system introduced by Fields and coworkers. In recent years, similar genetic assays have been developed in bacteria. We describe here several of these systems and highlight some potential applications of these technologies.

Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope.

Bordetella pertussis secretes an invasive adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain into the cytosol of eukaryotic target cells directly through the cytoplasmic membrane. We have shown previously that recombinant CyaA can be used to deliver viral CD8+ T cell epitopes to the MHC-class I presentation pathway to trigger specific CTL responses in vivo. In the present study, we show that mice immunized with a detoxified but still invasive CyaA carrying a CD8+ T cell epitope of OVA developed strong epitope-specific CTL responses, which kill tumor cells expressing this Ag. Treating mice with this recombinant molecule after the graft of melanoma cells expressing OVA induced a strong survival advantage compared with control animals. To our knowledge, this study represents the first demonstration that a nonreplicative and nontoxic vector carrying a single CTL epitope can stimulate efficient protective and therapeutic antitumor immunity.

Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway.

Among bacterial toxins, the adenylate cyclase toxin of Bordetella pertussis (CyaA) has a unique mechanism of entry that consists in the direct translocation of its catalytic domain across the plasma membrane of target cell, a mechanism supposed to be independent of any endocytic pathway. Here, we report that the CyaA toxin is delivered to the cytosolic pathway for MHC class I-restricted Ag presentation. Using peritoneal macrophages as APC, we show that the OVA 257-264 CD8+ epitope genetically inserted into a detoxified CyaA (CyaA-OVA E5) is presented to CD8+ T cells by a mechanism requiring 1) proteasome processing, 2) TAP, and 3) neosynthesis of MHC class I. We demonstrate that the presentation of CyaA-OVA E5, like the translocation of CyaA into eukaryotic cells, is dependent on extracellular Ca2+ and independent of vacuolar acidification. Moreover, inhibitors of the phagocytic and macropinocytic endocytic pathways do not affect the CyaA-OVA E5 presentation. The absence of specific cellular receptors for CyaA correlates with the ability of various APC to present the recombinant CyaA toxin, including dendritic cells, macrophages, splenocytes, and lymphoid tumoral lines. Taken together, our results show that the CyaA presentation pathway is not cell type specific and is unrelated to a defined type of endocytic mechanism. Thus, it represents a new and unconventional delivery of an exogenous Ag into the conventional cytosolic pathway.

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: implication for the in vivo delivery of CD8(+) T cell epitopes into antigen-presenting cells.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8(+) cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11-25 amino acids, corresponding to defined CD8(+) T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (-1 or -2) decreased or completely blocked (charge of -4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

A bacterial two-hybrid system based on a reconstituted signal transduction pathway.

We describe a bacterial two-hybrid system that allows an easy in vivo screening and selection of functional interactions between two proteins. This genetic test is based on the reconstitution, in an Escherichia coli cya strain, of a signal transduction pathway that takes advantage of the positive control exerted by cAMP. Two putative interacting proteins are genetically fused to two complementary fragments, T25 and T18, that constitute the catalytic domain of Bordetella pertussis adenylate cyclase. Association of the two-hybrid proteins results in functional complementation between T25 and T18 fragments and leads to cAMP synthesis. Cyclic AMP then triggers transcriptional activation of catabolic operons, such as lactose or maltose, that yield a characteristic phenotype. In this genetic test, the involvement of a signaling cascade offers the unique property that association between the hybrid proteins can be spatially separated from the transcriptional activation readout. This permits a versatile design of screening procedures either for ligands that bind to a given "bait," as in the classical yeast two-hybrid system, or for molecules or mutations that block a given interaction between two proteins of interest.

Characterization of DNA binding sites for the BvgA protein of Bordetella pertussis.

Expression of virulence-associated genes in Bordetella pertussis is under the control of the pleiotropic regulator BvgA. Although previous studies have identified recognition sequences for BvgA in several promoter regions, their structures have not been clearly characterized. We show that the BvgA binding sites within the bvgp(1) and cyaA promoters consist of inverted repeats and suggest that inverted-repeat motifs may represent the recognition elements for DNA-BvgA interaction.

Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis.

In Bordetella pertussis, transcription of virulence-associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal-transduction family. BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli. The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate. Using electrophoretic mobility-shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA-P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from -137 to -51, appears to be the target for BvgA-P binding, and probably contains multiple binding sites.

[Cloning and detailed mapping of the fra-ymt region of the Yersinia pestis pFra plasmid]. / Klonirovanie i detal'noe kartirovanie fra-ymt-oblasti plazmidy pFra Yersinia pestis.

The genetical libraries of the pFra plasmid of Yersinia pestis genes were obtained by insertion into the PstI, SalGI, EcoRI, XhoI restriction sites of the cosmid vector pHC79. The immunochemical analysis of the recombinant clones has revealed the clones synthesizing the antigen Fl (fraction I) and mouse toxin (Ymt--Yersinia pestis murine toxin). The restriction analysis of the plasmids from antigen synthesizing clones has permitted to construct the detailed physical map of the fra-ymt region of the pFra plasmid the size of 22 kb. The recombinant F1 positive clones of Escherichia coli are able to form at 37 degrees C the capsule-like structure peculiar for Yersinia pestis. The antigen F1 and the mouse toxin were isolated, purified and characterized. The antigen F1 is an 1-2 Md polymer containing a 16 kDa protein subunit. The mouse toxin a 240 kDa protein consisting of 61 kDa subunits. The nucleotide sequence of ymt gene has been defined.

[Specific proteolysis by fibrinolysin-coagulase from Yersinia pestis of Yersinia pseudotuberculosis outer membrane proteins coded by the Ca(2+)-dependence plasmid]. / Spetsificheskii proteoliz fibrinolizinokoagulazoi Yersinia pestis belkov vneshnei membrany, kodiruemykh plazmidoi Ca(2+)-zavisimosti v Yersinia pseudotuberculosis.

The pesticinogenicity 9.5 kb plasmid from Yersinia pestis strain EV76 has been marked by the kanamycin phosphotransferase gene inserted into PstI site and designated pP3. The obtained plasmid pP3 determines the synthesis of 45 kd pesticin, alpha and beta-forms of fibrinolysin coagulase (37 and 35 kd) and the 29, 19 and 13 kd proteins in Escherichia coli mini cells. When transferred into Yersinia pseudotuberculosis strain 6933 the plasmid causes the proteolysis of outer membrane proteins. The 150 kd protein is reduced to 138 kd, the 48.5 kd protein is reduced to 45 kd. The proteins secreted into the cultural medium (51 and 38 kd) are also cleaved. The proteolysis of the 150 kd protein was found to occur at the stage of secretion via the inner membrane. The purified fibrinolysin coagulase from Escherichia coli strain JM83 harbouring the plasmid pP3 induces the proteolysis in vitro of the isolated membrane proteins from Yersinia pseudotuberculosis strain 6953 similar to the proteolysis registered in vivo.