Influence of mutation in cj0183 and cj0588 genes for colonization abilities of Campylobacter jejuni in Caco-2 cells using confocal laser scanning microscope.

The cj0183 and cj0588 genes identified in the Campylobacter jejuni NCTC 11168 genome encode proteins with homology to virulence factors found in other bacteria. Previous studies showed that single mutation in the cj0183 gene does not affect adhesion of C. jejuni to the Caco-2 cell line whereas protein encoded by cj0588 is involved in adherence to the Caco-2 cells. In the presented study differences in invasion index were observed between mutants in both genes and single mutation of cj0588 in 81116 and 81-176 C. jejuni strains This fact indicates that Cj0183 protein might play some role in invasion of bacteria into host cells.

The first description of gastric Helicobacter in free-ranging wild boar (Sus scrofa) from Poland.

Specimens of gastric mucosa of 17 free-ranging wild boars (Sus scrofa) shot in the Central Poland during 2007/2008 hunting season were investigated for the presence of Helicobacter species. Histopathology, Helicobacter genus-specific 16S rRNA PCR, and DNA sequence analysis were employed. In PCR analysis the presence of Helicobacter's DNA was detected in one stomach. Obtained sequence analysis showed its relatedness to Helicobacter heilmannii type 2. In histopathology of the PCR-positive sample the presence of tightly coiled spiral bacteria was detected on the surface of the antral mucosa, in gastric pits and lumen of the upper parts of antral glands. Potential pathologic significance of the presence of Helicobacter in the stomach of free-ranging wild boars was obscured by the parasitic invasion-caused gastritis, and remains unknown.

Strategies of constructing recombinant vaccines.

Vaccines are the most effective prophylactic tool in veterinary medicine. Despite the great success of many vaccines used currently, there is still a constant need for their improvement. An ideal vaccine should contain a variety of immunogens, be safe and efficacious and induce broad humoral and cell-mediated immunity with one or, at most, two administrations given orally rather than by injection, and should be inexpensive. Traditional approaches include attenuated live vaccines, inactivated vaccines and subunit vaccines. Recently, scientific advances in molecular biology, immunology and bioinformatics, as well as the growing number of sequenced genomes of pathogens, have led to significant progress in respect to understanding virulence mechanisms at the molecular level. Genetic engineering has been applied to obtain recombinant bacterial and viral genomes in order to produce a modified and safe product useful in vaccine development. This article presents the progress and novel strategies used in creating new generation vaccines. It focuses on methods of searching for vaccine candidates to construction of vaccines based on recombinant DNA or proteins.

Resistance to antimicrobial agents of Campylobacter spp. strains isolated from animals in Poland.

A total of 69 Campylobacter jejuni and 16 Campylobacter coli strains isolated from chicken, dog and pig stool samples were characterized based on their resistance to five antimicrobial agents and on plasmid pTet profiles. Antimicrobials used in this study were: amoxicillin/clavulanic acid, ciprofloxacin, erythromycin, tetracycline and trimethoprim/sulfamethoxazole. Among the isolates studied, 91.7% were resistant to one or more antimicrobial agent. The highest level of resistance for the whole test group was to trimethoprim/sulfamethoxazole (57.6%), followed by ciprofloxacin (44.2%) and tetracycline (20%). All isolates were susceptible to amoxicillin/clavulanic acid. Strains isolated from chickens were susceptible to erythromycin. Few erythromycin-resistant strains were isolated from dogs and pigs (5.8%). C. coli strains exhibited a higher antibiotic resistance than C. jejuni strains, excluding resistance to trimethoprim/sulfamethoxazole. The pTet plasmid harboring the tet(O) gene was detected in 14 Campylobacter spp. strains. Our studies demonstrate that the majority (71.4%) of tetracycline-resistant isolates carry a plasmid-borne tet(O) gene, particularly strains for which the minimum inhibitory concentration (MIC) are > or = 256 microg/ml. In conclusion, we have found high-level trimethoprim/sulfamethoxazole, ciprofloxacin and tetracycline resistance in Polish strains isolated from different sources. This study has demonstrated that resistance of Campylobacter species differs depending on both the bacterial species and animal origins. All strains that displayed resistance to four antimicrobial agents were isolated from pigs. Localization of the tet(O) gene on either plasmid or chromosome was not found to be correlated with tetracycline resistance.

Predicting the properties of micro- and nanocomposites: from the microwhiskers to the bristled nano-centipedes.

The paper draws on the similarities between the well-known process of whiskerization of microfibres and the recent idea of bristled nanowires. The new method for evaluation of the effective elastic properties of such materials is suggested based on the model of four-component composition. This model assumes the transverse isotropy of continuum and predicts five elastic moduli and density as independent effective constants. An example of calculation of the constants for the particular materials is given. It shows the significant increase in the shear strength of composites with whiskerized or bristled fibres.

[Systemic fibrinolytic therapy using urokinase in central retinal artery occlusion. A case study]. / Systemische fibrinolytische Therapie mit Urokinase bei Zentralarterienverschluss der Netzhaut. Eine Fallstudie.

BACKGROUND AND OBJECTIVE: Systemic fibrinolysis has become an important therapeutical option in patients with thrombotic occlusion of coronary or pulmonary arteries. In view of the hemorrhagic risk systemic fibrinolytic therapy for retinal vessel occlusion has been discussed controversial. In the present case study results and complications of systemic fibrinolysis should be investigated in patients with central retinal artery occlusion. PATIENTS AND METHODS: From 1995 to 2002 a case series of 19 consecutive patients (8 female, 11 male, age: 63.2+/-14,3 years) with central retinal artery occlusion were treated by systemic application of urokinase using a standardized scheme. The latency from initial symptoms until the initiation of therapy and the medical history of the patients were documented. Visual acuity was determined on admission and before discharge and possible complications were documented. Additionally, screening investigations for genetic thrombophilia were performed. RESULTS: 15 patients showed an improvement of the visual acuity (79 %, 95 %-KI: 54 %-94 %). For 3 patients no improvement and for one patient a decrease of the visual acuity was determined. Hemorrhagic complications were observed in two patients (11 %, 95 %-KI: 1 %-33 %). As these minor bleedings were self-limiting the fibrinolytic therapy was discontinued only in one patient. As risk factors most commonly arterial hypertension (68 %) and smoking (26 %) were identified. In 4 patients a genetic thrombophilia was diagnosed. CONCLUSIONS: Considering the poor prognosis of central retinal artery occlusion and the disappointing results of conservative treatment, an improvement of the visual acuity in the absence of critical complications was observed with systemic fibrinolytic therapy in the presented case study. However, only controlled trials can provide proof for the effect of fibrinolysis versus spontaneous improvement.

What do we know about how humans cough?

We review the evidence that activation of the cerebral cortex can lead to movements of the vocal folds and possibly to cough. Electrical stimulation of the motor cortex can cause movements of the vocal folds and vocalizatioin, but cough has not been reported. The motor pathways are via the nucleus ambiguous and possibly the nucleus retroambuigualis in the brainstem. In humans, activation of the cerebral cortex by transcranial magnetic stimulation can cause motor potentials in the intrinsic laryngeal muscles and corresponding surface potentials. The relationships between the cortical sensation related to cough, the voluntary control of cough and the involvement of reflex pathways remain to be clarified.

Repeated exercise paired with "imperceptible" dead space loading does not alter VE of subsequent exercise in humans.

We employed an associative learning paradigm to test the hypothesis that exercise hyperpnea in humans arises from learned responses forged by prior experience. Twelve subjects undertook a "conditioning" and a "nonconditioning" session on separate days, with order of performance counterbalanced among subjects. In both sessions, subjects performed repeated bouts of 6 min of treadmill exercise, each separated by 5 min of rest. The only difference between sessions was that all the second-to-penultimate runs of the conditioning session were performed with added dead space in the breathing circuit. Cardiorespiratory responses during the first and last runs (the "control" and "test" runs) were compared for each session. Steady-state exercise end-tidal PCO(2) was significantly lower (P = 0.003) during test than during control runs for both sessions (dropping by 1.8 +/- 2 and 1.4 +/- 3 Torr during conditioning and nonconditioning sessions, respectively). This and all other test-control run differences tended to be greater during the first session performed regardless of session type. Our data provide no support for the hypothesis implicating associative learning processes in the ventilatory response to exercise in humans.

Central chemosensitivity and breathing asleep in unilateral medullary lesion patients: comparisons to animal data.

The rostro-ventrolateral medulla (RVLM) is a site of chemosensitivity in animals; such site(s) have not been defined in humans. We studied the effect of unilateral focal lesions in the rostrolateral medulla (RLM) of man, on the ventilatory CO(2) sensitivity and during awake and sleep breathing. Nine patients with RLM lesions (RLM group), and six with lesions elsewhere (non-RLM group) were studied. The ventilatory CO(2) sensitivity was lower in the RLM compared with the non-RLM group (mean (S.D.), RLM, 1.4 (0.9), non-RLM 3.0 (0.6) L min(-1) mmHg(-1)). In both groups resting breathing was normal. During sleep all RLM patients had frequent arousals, four had significant sleep disordered breathing (SDB), only one non-RLM patient had SDB. Our findings in humans resemble those in animals with focal RVLM lesions. This review provides evidence that in humans there is an area of chemosensitivity in the RLM. We propose that in humans, dorsal displacement of the RVLM area of chemosensitivity in animals, arises from development of the olive plus the consequences of the evolution of the cerebellum/inferior peduncle.

High strength stimulation of the vagus nerve in awake humans: a lack of cardiorespiratory effects.

UNLABELLED: Vagus nerve stimulation is used to reduce the frequency and intensity of seizures in patients with epilepsy. In the present study four such patients were studied while awake. We analyzed the physiological responses to vagus nerve stimulation over a broad range of tolerable stimulus parameters to identify vagal A-fiber threshold and to induce respiratory responses typical of C-fiber activation. A-fiber threshold was determined by increasing stimulation current until laryngeal motor A-fibers were excited (frequency=30 Hz). With A-fiber threshold established, C-fiber excitation was attempted with physiologically appropriate stimulus parameters (low frequency and high amplitude). RESULTS: A-fiber thresholds were established in all patients, threshold currents ranged between 0.5 and 1.5 mA. Stimulation at lower frequency (2-10 Hz) and higher amplitudes (2.75-3.75 mA) did not produce cardiorespiratory effects consistent with C-fiber activation. It is possible that such effects were not observed because vagal C-fibers were not excited, because C-fiber effects were masked by the 'wakeful drive' to breathe, or because epilepsy or the associated therapy had altered central processing of the vagal afferent inputs.

Identification of higher brain centres that may encode the cardiorespiratory response to exercise in humans.

1. Positron emission tomography (PET) was used to identify the neuroanatomical correlates underlying 'central command' during imagination of exercise under hypnosis, in order to uncouple central command from peripheral feedback. 2. Three cognitive conditions were used: condition I, imagination of freewheeling downhill on a bicycle (no change in heart rate, HR, or ventilation, V(I)): condition II, imagination of exercise, cycling uphill (increased HR by 12 % and V(I) by 30 % of the actual exercise response): condition III, volitionally driven hyperventilation to match that achieved in condition II (no change in HR). 3. Subtraction methodology created contrast A (II minus I) highlighting cerebral areas involved in the imagination of exercise and contrast B (III minus I) highlighting areas activated in the direct volitional control of breathing (n = 4 for both; 8 scans per subject). End-tidal P(CO(2)) (P(ET,CO(2))) was held constant throughout PET scanning. 4. In contrast A, significant activations were seen in the right dorso-lateral prefrontal cortex, supplementary motor areas (SMA), the right premotor area (PMA), superolateral sensorimotor areas, thalamus, and bilaterally in the cerebellum. In contrast B, significant activations were present in the SMA and in lateral sensorimotor cortical areas. The SMA/PMA, dorso-lateral prefrontal cortex and the cerebellum are concerned with volitional/motor control, including that of the respiratory muscles. 5. The neuroanatomical areas activated suggest that a significant component of the respiratory response to 'exercise', in the absence of both movement feedback and an increase in CO(2) production, can be generated by what appears to be a behavioural response.

Synthesis of 1-(3-aminopropyl)-4-phenyl-1,2,4-triazolin-5-one and 1-(3-aminopropyl)-3,4-diphenyl-1,2,4-triazolin-5-one.

The reaction of 4-phenyl-1,2,4-triazolin-5-one [Ia] and 3,4-diphenyl-1,2,4-triazolin-5-one [Ib] with 1-bromo-3-chloropropane was carried out. The obtained compounds [IIa, b] were subjected to the reaction with secondary amines and ethylenediamine with resulted in 1-(3-aminopropyl)-1,2,4-triazolin-5-one derivatives.

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by dynamic light scattering and time-resolved fluorescence anisotropy methods.

Cyclic AMP receptor protein (CRP) regulates the expression of more than 100 genes in Escherichia coli when complexed with cyclic AMP. Dynamic light scattering (DLS) and fluorescence decay anisotropy measurements of CRP were performed in solution, in the absence and presence of cAMP. We have also measured the effect of DNA sequences, including lac and gal promoter sequences, on the shape of CRP-DNA complexes. DLS measurements show that upon cAMP binding at low nucleotide concentration, the Stokes radius decreases from the value of 2.8 nm for apo-CRP to the value of 2.7 nm. At higher cAMP concentration, only a very small further decrease was detected. Fluorescence anisotropy decay measurements, with the use of CRP labeled at Cys-178 with 1,5-I-AENS, indicate that apo-CRP exhibits two rotational correlation times. The longer time, theta1 = 23.3 ns, corresponds to the overall motion of the protein, and the shorter time, theta2 = 1.4 ns, exhibits segmental mobility of the C-terminal domain of CRP. Binding of cAMP into CRP induced substantial increase of theta1 to the value of 30.7 ns, whereas theta2 remained unchanged. The DLS measurements indicate that the binding of CRP into a fragment of DNA possessing a sequence of lac promoter induces a larger increase in the Stokes radius of lac-CRP complex than in case of gal-CRP complex. Similarly, a higher change was detected in rotational correlation time, theta1, in the case of lac-CRP complex than in case of gal-CRP. Because the lac and gal promoters are characteristic for the two different classes of CRP-dependent promoters, one can expect that the observed differences in lac-CRP and gal-CRP complexes are important in activation of transcription in Escherichia coli.

Modulation of the corticospinal control of ventilation by changes in reflex respiratory drive.

We have determined whether changes in PCO(2) above and below eucapnia modulate the precision of the voluntary control of breathing. Twelve trained subjects performed a compensatory tracking task in which they had to maintain the position of a cursor (perturbed by a variable triangular forcing function) on a fixed target by breathing in and out of a spirometer (ventilatory tracking; at 10 l/min). Before each task, subjects hyperventilated for 5 min, and the end-tidal PCO(2) (PET(CO(2))) was controlled; tracking was then performed separately at hypocapnia, eucapnia, and hypercapnia (PET(CO(2)) approximately 25, 37, and 43 Torr, respectively). Ventilatory tracking error was unchanged during hypocapnia (P > 0.05) but was significantly worse during hypercapnia (P < 0.003), compared with eucapnia; arm tracking error, performed as a control, was not significantly affected by PET(CO(2)) (P > 0. 05). In conclusion, ventilatory tracking performance is unaffected by the eucapnic PCO(2). From this, we suggest that resting breathing in awake humans may be independent of chemical drives and of the prevailing PCO(2).

Unilateral focal lesions in the rostrolateral medulla influence chemosensitivity and breathing measured during wakefulness, sleep, and exercise.

OBJECTIVES: The rostrolateral medulla (RLM) has been identified in animals as an important site of chemosensitivity; in humans such site(s) have not been defined. The aim of this study was to investigate the physiological implications of unilateral lesions in the lower brainstem on the control of breathing. METHODS: In 15 patients breathing was measured awake at rest, asleep, during exercise, and during CO(2) stimulation. The lesions were located clinically and by MRI; in nine patients they involved the RLM (RLM group), in six they were in the pons, cerebellum, or medial medulla (Non-RLM group). All RLM group patients, and three non-RLM group patients had ipsilateral Horner's syndrome. RESULTS: Six of the RLM group had a ventilatory sensitivity to inhaled CO(2) (V/P(ET) CO(2)) below normal (group A: V/P(ET) CO(2), mean, 0.87; range 0.3-1.4 l. min(-1)/mm Hg). It was normal in all of the non-RLM group (group B: V/P(ET) CO(2), mean, 3.0; range, 2.6-3.9 min(-1)/mmHg). There was no significant difference in breathing between groups during relaxed wakefulness (V, group A: 7.44 (SD 2.5) l.min(-1); group B: 6.02 (SD 1.3) l.min(-1); P(ET) CO(2), group A: 41.0 (SD 4.2) mm g; group B: 38.3 (SD2.0) mm Hg) or during exercise (V/VO(2): group A: 21 (SD 6. 0) l.min(-1)/l.min(-1); group B: 24 (SD 7.3) l.min(-1)/l.min(-1)). During sleep, all group A had fragmented sleep compared with only one patient in group B (group A: arousals, range 13 to > 60 events/hour); moreover, in group A there was a high incidence of obstructive sleep apnoea associated with hypoxaemia. CONCLUSION: Patients with unilateral RLM lesions require monitoring during sleep to diagnose any sleep apnoea. The finding that unilateral RLM lesions reduce ventilatory sensitivity to inhaled CO(2) is consistent with animal studies. The reduced chemosensitivity had a minimal effect on breathing awake at rest or during exercise.

Purification and N-terminal amino acid sequence of sheep neutrophil cathepsin G and elastase.

Sheep cathepsin G (CG) and neutrophil elastase (NE) were isolated from a crude leukocyte membrane preparation by elastin-Sepharose 4B and CM-Sepharose 4B chromatography, followed by native preparative PAGE. The N-termini of CG and NE were sequenced to 24 and 20 residues, showing 96 and 85% identity with human CG and NE, respectively. During SDS-PAGE, sheep CG and NE migrated parallel to human CG and NE and have apparent molecular masses of 28 and 26 kDa, respectively. Following incubation of sheep CG and NE with human alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor, complexes with apparent molecular masses of 89 and 81 kDa respectively were observed by SDS-PAGE. Polyclonal antibodies to human CG and NE cross-reacted with purified sheep CG and NE, respectively. These results indicate that sheep neutrophils contain CG and elastase that are analogous to human CG and NE in terms of molecular mass, reactivity with endogenous inhibitors, immunocross-reactivity, and N-terminal sequence.

Cardiorespiratory variables and sensation during stimulation of the left vagus in patients with epilepsy.

We studied physiological and sensory effects of left cervical vagal stimulation in six adult patients receiving this stimulation as adjunctive therapy for intractable epilepsy. Stimulus strength varied among subjects from 0.1 to 2.1 microCoulomb (microC) per pulse, delivered in trains of 30-45 s at frequencies from 20 to 30 Hz; these stimulation parameters were standard in a North American study. The stimulation produced no systematic changes in ECG, arterial pressure, breathing frequency tidal volume or end-expiratory volume. Five subjects experienced hoarseness during stimulation. Three subjects with high stimulus strength (0.9-2.1 microC) recalled shortness of breath during stimulation when exercising; these sensations were seldom present during stimulation at rest. No subjects reported the thoracic burning sensation or cough previously reported with chemical stimulation of pulmonary C fibers. Four of six subjects (all those receiving stimuli at or above 0.6 microC) experienced a substantial reduction in monthly seizure occurrence at the settings used in our studies. Although animal models of epilepsy suggest that C fibers are the most important fibers mediating the anti-seizure effect of vagal stimulation, our present findings suggest that the therapeutic stimulus activated A fibers (evidenced by laryngeal effects) but was not strong enough to activate B or C fibers.

Cortical and subcortical control of tongue movement in humans: a functional neuroimaging study using fMRI.

We have used voluntary tongue contraction to test whether we can image activation of the hypoglossal nuclei within the human brain stem by using functional magnetic resonance imaging (fMRI). Functional images of the whole brain were acquired in eight subjects by using T2-weighted echo planar imaging (blood oxygen level development) every 6.2 s. Sequences of images were acquired during 12 periods of 31-s "isometric" rhythmic tongue contraction alternated with 12 periods of 31-s tongue relaxation. Noise arising from cardiac- and respiratory-related movement was removed either by filtration (high pass; cutoff 120 s) or by inclusion in the statistical analysis as confounding effects of no interest. For the group, tongue contraction was associated with significant signal increases (P < 0.05 corrected for multiple comparisons) in the sensorimotor cortex, supplementary motor area, operculum, insula, thalamus, and cerebellum. For the group and for six of eight individuals, significant signal increases were also seen within the medulla (P < 0.001, predefined region of interest with no correction for multiple comparisons); this signal is most likely to reflect neuronal activation associated with the hypoglossal motor nuclei. The data demonstrate that fMRI can be used to detect, simultaneously, the cerebral and brain stem control of tongue movement.

Does the motor cortical control of the diaphragm 'bypass' the brain stem respiratory centres in man?

In humans, cortico-motor excitation of the diaphragm may act directly on the phrenic motor nucleus via the cortico-spinal tract 'bypassing' brain stem respiratory centres (RC); alternatively, or in addition, this control may be indirect via the RC and bulbo-spinal paths. To investigate this, we stimulated the motor cortex using transcranial magnetic stimulation (TMS) in six subjects at end-expiration (diaphragm relaxed) and during voluntary inspiration. The sizes of the evoked compound action potentials in the diaphragm and also, as a control, in the thumb were no different whether TMS was delivered during normocapnia or during hypocapnia (PET(CO2) = 25 mmHg) when, presumably, the respiratory 'oscillator' was silent. In a further six subjects, TMS was performed during relaxed spontaneous breathing at three different points in the respiratory cycle. No perturbations in respiratory pattern (either tidal volume or respiratory timing) were seen. Thus we have been unable to demonstrate that the cortico-motor excitation of the diaphragm acts via the brain stem RC.