In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6×His tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 Μg/ml ampicillin at 37°C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.
During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites. However, purified HPr-Ser-P protein from Bacillus subtilis was shown to slightly increase the DNA-binding capacity of the CcpA protein. It was also observed that CcpA bound to the cre box forms an apparently more stable complex than that resulting from unspecific binding. Competition gel retardation assay performed on DNA sequences from two PEP:PTS regions demonstrated that the ybhE, bglS, rheB, yebE, ptcB and yecA genes situated in these regions are most probably directly regulated by CcpA.
In the gastrointestinal tract (GIT), adhesion is a prerequisite for bacterial colonization. Lactococci can be used in functional food (probiotics) and health-related applications (mucosal vaccines, therapeutic drug delivery), both potentially involving adhesive properties. A candidate lactic acid bacterium for influenza antigen delivery through the GIT should display the ability to adhere. The present work probes the interactions between Lactococcus lactis and mucins using pig gastric mucin (PGM) as a model. Two strains were used for the optimization of the screening method for adhesion: L. lactis subsp. cremoris IBB477 persistent in the GIT of germ-free rats, and the low-adhering control strain MG1820. High adhesion to bare and mucin-coated polystyrene of IBB477 in comparison with MG1820 was observed. We searched for genetic determinants potentially involved in the adhesion/muco-adhesion of IBB477, identifying two such genes: prtP and a gene coding for a protein with MUB and MucBP domains. Based on its persistence in the GIT and adhesive properties, L. lactis IBB477 is a candidate carrier strain for expression of influenza haemagglutinin (HA) protein for induction of mucosal immune response.
Gastrooesophageal reflux disease is the regurgitation of stomach contents into the esophagus, which causes troublesome symptoms or complications for the patient. Before starting the treatment, it is always necessary to objectively confirm gastroesophageal reflux disease, especially in correlation with ENT symptoms, as extra esophageal complications. In diagnostics, the "gold standard" is a 24-hour impedance-pH supplemented with endoscopy. Treatment without objective confirmation of the disease is not recommended, the more so that non-acid gas proximal reflux, detectable only in the MIIpH test, causes the greatest number of laryngological complications. It is important to confirm the coexistence of clinical symptoms of GERD with ESS. Considering the time of treating the disease and its consequences, it is worthwhile to be cautious and careful with the diagnosis of the disease, and the treatment should be carried out for a long time in relation to the recommendation, preferably in cooperation with an ENT specialist and gastroenterologist. The greatest therapeutic effectiveness is achieved by combining PPI with itopride while maintaining the appropriate doses of drugs and observing a sufficiently long duration of treatment, while maintaining the correct dose reduction and drug discontinuation regimen. In case of failure of pharmacological treatment, antireflux surgery should be take into consideration.
Choroba refluksowa przełyku to zarzucanie treści żołądkowej do przełyku, powodujące kłopotliwe dla pacjenta objawy lub powikłania. Przed włączeniem leczenia należy zawsze obiektywnie potwierdzić tę chorobę, zwłaszcza w korelacji z dolegliwościami laryngologicznymi jako powikłaniami pozaprzełykowymi. W diagnostyce „złotym standardem” jest 24-godzinna impedancja-pH, uzupełniona endoskopią. Nie zaleca się leczenia bez obiektywnego potwierdzenia choroby, tym bardziej, że najwięcej powikłań laryngologicznych powoduje refluks niekwaśny gazowy proksymalny, możliwy do detekcji wyłącznie w badaniu MII-pH. Ważne jest potwierdzenie współwystępowania wraz z zespołami pozaprzełykowymi objawów klinicznych GERD (ang. Gastroesophgaeal Reflux Disease). Mając na uwadze czas leczenia choroby i jej konsekwencji, warto rozważnie i ostrożnie podchodzić do rozpoznania choroby, a leczenie powinno być odpowiednio długo prowadzone, w odniesieniu do rekomendacji, najlepiej przy współpracy specjalisty laryngologa i gastroenterologa. Największą efektywność terapeutyczną uzyskujemy poprzez połączenie inhibitorów pompy protonowej (PPI) z itoprydem przy zachowaniu odpowiednich dawek leków oraz przestrzegając odpowiednio długiego czasu leczenia z zachowaniem prawidłowego schematu redukcji dawek i odstawiania leków. W przypadku nieskuteczności leczenia zachowawczego, należy rozważyć leczenie chirurgiczne antyrefluksowe.
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