In the study presented here, identification, purification, and partial characterization of a hemin-regulated protein in Prevotella nigrescens were carried out. The results of this study confirm that the availability of hemin influences the expression of a selected membrane protein as well as the growth rate of P. nigrescens ATCC 33563. The 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin-binding protein from P. nigrescens. Disulfide bonds were not present in this protein, and N'-terminal amino acid sequence analysis revealed that this protein belongs to a new, so far undescribed protein. The 50 kDa protein was found to be rich in hydrophilic amino acids, with glycine comprising approximately 60% of the total amino acids. The study described here is the first to identify, purify, and biochemically characterize a putative hemin-binding protein from P. nigrescens. Work is in progress to further characterize the molecular structure of this protein.
Amino Acids ; Glycine ; Hemin ; Membrane Proteins ; Molecular Structure ; Prevotella nigrescens* ; Prevotella* ; Sequence Analysis, Protein

This study was performed to observe the antibacterial effect of polyphosphate (polyP) with various chain lengths (P3~P75) on virulent, invasive strains of P. gingivalis A7A1-28 and W50, and multidrug resistant E. faecalis ATCC29212. P. gingivalis strains were grown in brain-heart infusion broth (BHI) containing hemin and vitamin K with or without polyP. PolyP was added at the very beginning of the culture or during the exponential growth phase of the culture. Inhibition of the growth of P. gingivalis was determined by measuring the absorbancy at 540nm of the grown cells. Viable cell counts of the culture and release of intracellular nucleotide from P. gingivalis were measured. E. faecalis was grown in plain BHI with antibiotics alone or in combination with polyP(calgon; 0.1~1.0%) and the bacterial absorbancy was measured. The overall results suggest that polyP has a strong antibacterial effect on the growth of the virulent strains of P. gingivalis and the antibacterial activity of polyP seems largely bactericidal, accompanying bacteriolysis in which chelation phenomenon is not involved. Although polyP does not exert antibacterial activity against E. faecalis, it appears to increase antibacterial effect of erythromycin and tetracycline on the bacterium. Therefore, polyP alone or in combination with antibiotics may be developed as a candidate for the agent controlling oral infections including endodontic infection.
Anti-Bacterial Agents ; Bacteria* ; Bacteriolysis ; Cell Count ; Erythromycin ; Hemin ; Polyps ; Tetracycline ; Vitamin K

Hemin blocked lipid peroxidations induced by either ascorbate/FeSO4, a metal-catalyzed oxidation system, or 2,2'-azobis-2-amidino-propane hydrochloride (ABAP) which produces peroxy radicals at constant rates. Hemin at very low micromolar concentrations strongly inhibited the ascorbate/FeSO4-induced peroxidation of rat liver phopholipids, soybean phosphatidylcholine and arachidonic acid, and this inhibition was also evident with the use of ABAP, although much higher concentrations of hemin were required than those for the inhibition of ascorbate/FeSO4-induced lipid peroxidation. However, hemoproteins such as hemoglobin, myoglobin and cytochrome C did not show any significant effect on this lipid peroxidation. Hemopexin and albumin abolished the inhibitory action of hemin. During incubation with ascorbate/FeSO4 or ABAP, hemin underwent a change in its absorption spectrum, resulting in a progressive decrease in the peak height of the characteristic absorption band at 385 nm. The above results suggest that hemin may act as an important antioxidant in vivo, protecting lipids from the peroxidative damage.
Absorption ; Animals ; Arachidonic Acid ; Cytochromes c ; Hemin* ; Hemopexin ; Lipid Peroxidation* ; Liver ; Myoglobin ; Phosphatidylcholines ; Rats ; Soybeans

A 34-year old woman visited the hospital complaining severe general pain which had onset on the way of improvement of sore throat, cough with sputum as symptoms of acute upper respiratory infection for 3 days. The facts that her younger sister also had a history of porphyria and the color of the patient's urine changed to dark black after it had exposed to sunlight made us to rule out porphyria strongly. Therefore, we measured the level of delta-ALA and porphobilinogen in the collected urine during 24 hours, and confirmed her diagnosis as acute intermittent porphyria. The SIADH was complicated and the sleep disturbance, disorientation and hallucination onset during the hospital days. She had taken high dose dextrose IV and hematin IV therapy for porphyria and improved gradually. Therefore, authors et al. report a case of acute intermittent porphyria with various clinical symptoms on the way of treatment of upper respiratory infection as well as review the previous literatures.
Cough ; Diagnosis ; Female ; Glucose ; Hallucinations ; Hemin ; Humans ; Inappropriate ADH Syndrome ; Pharyngitis ; Porphobilinogen ; Porphyria, Acute Intermittent ; Porphyrias* ; Siblings ; Sputum ; Sunlight

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain DH5alpha, and screened for recombinant clones with heminbinding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa heminbinding protein.
Agar ; Clone Cells ; DNA ; Escherichia coli ; Genomic Library ; Hemin ; Humans ; Iron ; Periodontal Diseases ; Phenotype ; Plasmids ; Porphyromonas gingivalis* ; Porphyromonas* ; RNA

There are estimated to be about 700 species of bacteria in the oral cavity. Based on epidemiological investigations, some of these strains have been proposed as the pathogens responsible for oral diseases such as dental caries, gingivitis and periodontitis. Since electrolyzed hydrogen-rich water has been shown to have beneficial effects on human immunity, its use has increased. In our study, the antibacterial activity of hydrogen-rich water for oralagainst bacteria associated with oral disease was evaluated. The bacterial strains Streptococcus mutans, Fusobacterium nucleatum, Porphyromonas gingivalis and Tannerella forsythia were cultured in specific growth medium. S. mutans, F. nucleatum and P. gingivalis were soaked to thein both hydrogen water and tap water for 30 sec and then inoculated onto mitis-salivarius agar and brain heart infusion agar including supplemented withvitamin K and hemin, respectively. The numbers of bacterial colonies were then measured after cultivation for 48 hours. In the case of T. forsythia, which does not grow well on agar plates, inoculations into modified new oral spirochete (NOS) broth were performed and growth curve analysis was undertaken every day with a spectrophotometer. Hydrogen water showed antibacterial activity against all four bacterial strains in comparison with tap-water. We conclude from this that hydrogen water may have a positive impact on oral hygiene by helping to remove cariogenic bacteria and periodontopathogens.
Agar ; Bacteria ; Brain ; Dental Caries ; Forsythia ; Fusobacterium nucleatum ; Gingivitis ; Heart ; Hemin ; Humans ; Hydrogen ; Mouth ; Oral Hygiene ; Periodontitis ; Porphyromonas gingivalis ; Spirochaetales ; Streptococcus mutans ; Water

Porphyromonas gingivalis is strongly implicated in the pathogenesis of adult periodontitis, the major cause of tooth loss in adults. Use of an antibacterial agent controlling P. gingivalis as a periodontal therapeutic agent has been rationalized. The present study was performed to observe the antibacterial effect of inorganic polyphosphates (polyP) on P. gingivalis. P. gingivalis 2561 was grown in half-strength brain-heart infusion broth containing hemin and vitamin K with or without polyP. Minimal inhibitory concentration (MIC) of polyP with various chain lengths was determined by measuring the absorbance of the grown cells at 540 nm. MIC of polyP for the bacterium was determined to be 0.05%. The effect of polyP with a chain length of 75 (polyP 75) was further examined. PolyP 75 added to the growing culture of P. gingivalis at its exponential phase was as effective in inhibiting the growth of P. gingivalis as polyP 75 added at the very beginning of the culture. More than 99% of the cells lost their viability determined by viable cell count when polyP 75 was added to the culture of growing P. gingivalis at the concentration of 0.06%, suggesting that polyP 75 has a bactericidal effect on the bacterium. Intracellular nucleotide release from the cells was increased by approx. 20% in the presence of polyP 75 but was not reversed by the addition of divalent cations like Ca++ and Mg++. Under the transmission electron microscope, only a small number of the growing P. gingivalis cells were actually lysed. However, the majority of the cells appeared to be atypical in their shape, demonstrating accumulation of highly electron-dense granules and bodies of condensed nucleic acid-like material in the cytoplasm. In the presence of polyP 75, the protein profile of P. gingivalis was changed as determined by SDS-polyacrylamide gel electrophoresis and immunoblot, and the proteolytic activity of the bacterium demostrated on the zymograms was decreased. The overall results suggest that polyP have a strong bactericidal activity against P. gingivalis in which lysis in relation to chelation may not play the major role but unknown mechanism that possibly affects the viability of the bacterium may be involved. PolyP may be used as an agent for prevention and treatment of periodontitis.
Adult ; Cations, Divalent ; Cell Count ; Chronic Periodontitis ; Cytoplasm ; Electrophoresis ; Hemin ; Humans ; Periodontitis ; Polyphosphates* ; Polyps ; Porphyromonas gingivalis* ; Porphyromonas* ; Tooth Loss ; Vitamin K

Recently, small colony variants (SCVs) of Staphylococcus aureus causing fatal infections are increasing, but rarely reported in Korea. S. aureus, SCVs are slow growing subpopulation that cause persistent and relapsing infections. S. aureus, SCVs are frequently auxotrophic for hemin, menadione, and CO2, and are often disrupted in their electron transport activity. With S. aureus, SCVs virulence is altered by a decrease in -toxin production and susceptibility to various antibiotics, allowing their intracellular survival. We isolated S. aureus, SCVs from the sputum of a 67 year old male with pneumonia, chronic renal failure with hemodialysis and preventive antibiotic therapy. Because S. aureus, SCVs are easily missed or misdiagnosed as normal flora in routine culture due to their atypical growth behavior and biochemical reaction, the correct identification is very important, especially when no bacteria or unusual bacteria are found in patients with persistent or relapsing infections with long term antibiotic therapy.
Aged ; Anti-Bacterial Agents ; Bacteria ; Electron Transport ; Hemin ; Humans ; Kidney Failure, Chronic* ; Korea ; Male ; Pneumonia ; Renal Dialysis ; Sputum* ; Staphylococcus aureus* ; Staphylococcus* ; Virulence ; Vitamin K 3

OBJECTIVE: Patient' desire of transfusion free surgery has been increasing due to blood transfusion risks. We analyzed the perioperative parameters and perioperative management of transfusion free surgical treatment in Soonchunhyang University Seoul Hospital. METHODS: Operation quantity and blood unstoring count from blood bank between 2000 and 2012 were collected from chronological records. Perioperative parameters including preoperative hemoglobin level, postoperative hemoglobin level, and lowest hemoglobin level were collected from medical records. Perioperative blood management such as acute normovolemic hemodilution, intraoperative blood cell salvage, or hematinic agents and complication were assessed. RESULTS: A total of 3,088 patients underwent transfusion free surgery at Soonchunhyang University Seoul Hospital between 2000 and 2012. Postoperative hemoglobin level <5.0 g/dL were 33 patients. Four patients expired at postoperative period with serious perioperative complications. Average of expired patient's hemoglobin was 3.22 g/dL and overall mortality was 0.12%. Operation was increased as years go by. The amount of blood use bank wasn't increased in general patients with transfusion. CONCLUSION: Careful perioperative blood management for transfusion free surgical treatment was responsible for safety and results in good clinical outcomes. Overall transfusion rate was decreased in spite of increasing operation quantity.
Blood Banks ; Blood Transfusion ; Bloodless Medical and Surgical Procedures ; Hemin ; Hemodilution ; Humans ; Korea ; Medical Records ; Mortality ; Operative Blood Salvage ; Perioperative Care ; Postoperative Period ; Seoul

BACKGROUND: Staphylococcus aureus and Group A beta-hemolytic Streptococci are the etiologic agents most commonly associated with cellulitis, but many other bacteria have also been shown to cause this condition. The positive bacterial culture rate is the most important factor in the treatment of cellulitis. However, the positive bacterial culture rate in the commonly used media, tends to be quite low. OBJECTIVE: The principal objective of this study was to improve the positive culture rate in cellulitis patients by using a new enriched broth. METHODS: Brewer modified thioglycollate medium (BTM) and Columbia broth (CB), both of which are widely utilized in clinical bacteriology for enriched growth, were compared with several novel enriched broths. These new enriched broths were mixtures of BTM-CB broth and added growth supplement factors. They included BTM-CB (BC), Modified BTM-CB (MBC) and supplement VX-BTM-CB (VXBC). MBC media included several growth supplements, such as hemin, vitamin K1, VX supplement, and Campylobacter growth supplement. Strains utilized in this study were common pathogens (Staphylococcus aureus, Streptococcus pyogenes, et al.), anaerobes, fastidious pathogens (Bacteroides fragilis, Campylobacter jejuni, Prevotella melaninogenica), uncommon pathogens (Actinobacter baumannii, Enterococcus faecalis, Streptococcus agalactiae). Positive culture rates were evaluated in each medium and measured via spectrophotometry at 660 nm. RESULTS: In vitro, all strains used in this study grew more quickly and densely in MBC media. CONCLUSION: Our results suggest that MBC media in a new enriched broth may improve bacterial culture rates in cellulitis patients. It will be necessary to study the efficacy of the MBC media in the culturing etiologic agents from tissues of cellulitis patients.
Bacteria ; Bacteriology ; Campylobacter ; Campylobacter jejuni ; Cellulitis ; Enterococcus faecalis ; Hemin ; Humans ; Organothiophosphorus Compounds ; Prevotella ; Spectrophotometry ; Staphylococcus aureus ; Streptococcus ; Streptococcus pyogenes ; Vitamin K 1