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

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

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

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

OBJECTIVES: Dental plaque is composed of 700 bacterial species. It is known that some oral microorganisms produce porphyrin, and thus, they emit red fluorescence when illuminated with blue light at a specific wavelength of <410 nm. Porphyromonas gingivalis belongs to the genus Porphyromonas, which is characterized by the production of porphyrin. The aim of this study was to evaluate red fluorescence emission of some oral microorganisms interacting with P. gingivalis. METHODS: Five bacterial strains (P. gingivalis, Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, and Fusobacterium nucleatum) were used for this study. Tryptic soy agar medium supplemented with hemin, vitamin K3, and sheep blood was used as a growth medium. The fluorescence emission of bacterial colonies was evaluated under 405 nm-wavelength blue light using a Quantitative Light-induced Fluorescence Digital (QLF-D) camera system. Each bacterium was cultured alone and co-cultured in close proximity with P. gingivalis. The red/green (R/G) ratio of fluorescence image was calculated and the differences of R/G ratio according to each growth condition were compared using the Mann-Whitney test (P<0.05). RESULTS: Single cultured S. mutans, L. casei and A. naeslundii colonies emitted red fluorescence (R/G ratio=2.15±0.06, 4.31±0.17, 5.52±1.29, respectively). Fusobacterium nucleatum colonies emitted green fluorescence (R/G ratio=1.36±0.06). The R/G ratios of A. naeslundii and F. nucleatum were increased when P. gingivalis was co-cultured with each bacterium (P<0.05). In contrast, the R/G ratios of S. mutans and L. casei were decreased when P. gingivalis was co-cultured with each bacterium (P=0.002, 0.003). CONCLUSIONS: This study confirmed that P. gingivalis could affect the red fluorescence of other oral bacteria under 405 nm-wavelength blue light. Our findings concluded that P. gingivalis has an important role for red fluorescence emission of dental biofilm.
Actinomyces ; Agar ; Bacteria* ; Biofilms ; Dental Plaque ; Fluorescence* ; Fusobacterium ; Fusobacterium nucleatum ; Hemin ; Lactobacillus casei ; Porphyromonas gingivalis* ; Porphyromonas* ; Sheep ; Streptococcus mutans ; Vitamin K 3

The results of this study confirm that the availability of hemin influences the expression of selected membrane proteins of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is hemin regulated was identified and purified in P. gingivalis. A strong hemin-binding function was found by LDS-PAGE and TMBZ staining when P. gingivalis cells were grown under hemin-limited conditions. A 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin binding protein from P. intermedia and P. nigrescens, respectively. N-terminal amino acid sequence analysis of CNBr-digested 24 kDa hemin binding protein from P. gingivalis revealed that this protein belongs to a new, so far undescribed hemin-binding class of proteins. N-terminal amino acid sequence of a 50 kDa putative hemin binding protein from P. intermedia was identical with Enolase from Streptococcus intermedia. Work is in progress to further characterize the molecular structure of these proteins.
Amino Acid Sequence ; Carrier Proteins ; Hemin ; Membrane Proteins ; Molecular Structure ; Phosphopyruvate Hydratase ; Porphyromonas gingivalis* ; Porphyromonas* ; Prevotella intermedia* ; Prevotella nigrescens* ; Prevotella* ; Sequence Analysis, Protein ; Streptococcus

OBJECTIVETo study the effect of endogenous carbon monoxide (CO) on the smooth muscle function of the dog penile corpus cavernosum in vitro.

METHODSTissue bioassay was used to measure the corpus cavernosum muscle contraction and relaxation. The production of CO was induced in the corpus cavernosum smooth muscle, and the effect of CO on the penile corpus cavernosum smooth muscle pre-contracted by phenylephrine (PE) was determined.

RESULTSChlorinous hemoglobin could relax the smooth muscle stripes pre-contracted by 10 micromol/L PE. A dose-dependent relaxation was observed. The relaxation responses by 10 -100 micromol/L chlorinous hemoglobin were significant compared with the control group (P < 0. 01). The pretreatment of the muscle stripes with ZnPP-IX or methylthioninium significantly reduced the relaxing effect of chlorinous hemoglobin (P < 0.01).

CONCLUSIONThe relaxing effect of endogenous CO on the smooth muscle of the penile corpus cavernosum depends on the concentration of endogenous CO. The underlying mechanism may involve the pathway from CO to cGMP production.

Animals ; Carbon Monoxide ; physiology ; Dogs ; Dose-Response Relationship, Drug ; Hemin ; pharmacology ; In Vitro Techniques ; Male ; Muscle, Smooth ; drug effects ; physiology ; Penile Erection ; drug effects ; physiology ; Penis ; drug effects ; physiology

OBJECTIVETo identify hemin-induced gene expression in K562 cells.

METHODSPoly A(+) RNAs were isolated from hemin-induced (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNAs were synthesized by reverse transcription. The forward subtracted cDNA library was constructed by using suppression subtractive hybridization (SSH) techniques. The recombinant plasmids were extracted and the positive clones were identified by EcoR I digestion after the amplification and screening of the library. The inserts were amplified by PCR. The upregulated cDNA transcripts were identified by reverse dot blot hybridization, DNA sequencing and homology analysis with GenBank database "blast" respectively.

RESULTSFifteen upregulated clones were identified and most of them were homologous to the mRNA sequences of protein with known function, including globin epsilon1, glutathione S-transferase like glutathione transferase Omega (GSTTLp28), selenoprotein X1 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin light polypeptide, globin A gamma, RAD 51 homolog C(RAD51C), ferritin heavy polypeptide, X-box binding protein (XBP1). A part of the hemin-induced cDNA clones exhibited sequence similarities to that of the GenBank registered mRNA with unknown function of their expressed proteins, including the cDNA clones of DKFZp434I116, hypothetical protein HSPC014 and NOL1R2 proteins.

CONCLUSIONSHemin mainly induces the genes expression related to erythroid differentiation, protein synthesis and metabolism in K562 cell. There results provide comprehensive information useful for the differential gene expression in hemin-induced erythroid differentiation and for further function study of genes involved in hematopoiesis.

Blotting, Northern ; DNA, Complementary ; drug effects ; genetics ; Gene Expression ; drug effects ; Hemin ; pharmacology ; Humans ; K562 Cells ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction

AIMTo investigate the expression of heme oxygenase-1 gene and production of endogenous carbon monoxide in the rat lung tissue at different time points of chronic hypoxic pulmonary hypertension and the effect of hemin, an inducer of heme oxygenase, on the expression of HO-1 gene and production of endogenous carbon monoxide and pulmonary hypertension.

METHODSWe recreated a rat model of hypoxic pulmonary hypertension by intermittent normal pressure hypoxia (10% O2). The following assays were carried out: Reverse transcriptase polymerase chain reaction (RT-PCR) were performed to determine the level of HO-1 mRNA in rat lung tissue, double wave length spectrophotometry was used to evaluate the quantity of COHb in arterial blood, cardiac catheterization was used to measure the right ventricular systolic pressure (RVSP) and HE staining was performed in dissected lung tissue to observe the pathologic changes of the intra-acinar pulmonary arteries(IAPA).

RESULTS(DT here was low level of HO-1 mRNA in normal rat lung tissue, but the level of HO-1 mRNA increased by 2-4 times in the lung tissue of hypoxic rats (P < 0.01). The quantity of COHb was 2-3 times as those of control group (P < 0.01 or P < 0.05). These were accompanied by the increase of RVSP and the thickness of IAPA. (2) Hemin could maintain the HO-1 mRNA and COHb in the hypoxic rat lung tissue at a high level, and partially suppressed the increase of rat RVSP, ameliorated the pathologic changes of IAPA.

CONCLUSIONThe upregulation of the expression of HO-1 gene and production of CO in the rat lung of hypoxic pulmonary hypertension plays a role of inhibition in the development of hypoxic pulmonary hypertension. Hemin has a therapeutic effect on hypoxic pulmonary hypertension.

Animals ; Carbon Monoxide ; metabolism ; Heme Oxygenase (Decyclizing) ; metabolism ; Hemin ; pharmacology ; Hypertension, Pulmonary ; metabolism ; pathology ; physiopathology ; Hypoxia ; metabolism ; pathology ; Male ; Pulmonary Artery ; metabolism ; physiopathology ; Rats ; Rats, Wistar