Inhibition of group A streptococcal infection by Melaleuca alternifolia (tea tree) oil concentrate in the murine model.

AIMS: To investigate the effect of a water-soluble Melaleuca alternifolia concentrate (MAC) on group A streptococcus (GAS; Streptococcus pyogenes)-induced necrotizing fasciitis. METHODS AND RESULTS: MAC pretreatment (1% and 2% v/v) was able to protect mice from GAS infection in an air pouch model. GAS-induced mouse death and skin injury were inhibited dose dependently by MAC. Administration of MAC at 6 h post-GAS infection partially delayed mouse death. Surveys of the exudates of the air pouch of MAC-treated mice revealed that the survival of infiltrating cells was prolonged, the bacteria were eliminated, and the production of inflammatory cytokines was inhibited. MAC could directly inhibit the growth of GAS in vitro, and the minimal inhibitory concentration (MIC) of MAC for GAS was determined as 0.05% v/v using the time-kill assay. Furthermore, a sub-MIC dose of MAC not only enhanced the bactericidal activity of RAW264.7 macrophage cells against GAS but also increased susceptibility of GAS for blood clearance. CONCLUSIONS: These results suggest that MAC may inhibit GAS-induced skin damage and mouse death by directly inhibiting GAS growth and enhancing the bactericidal activity of macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide scientific data on the use of MAC for the treatment of GAS-induced necrotizing fasciitis in the murine model.

The genomic structure of SYCP3, a meiosis-specific gene encoding a protein of the chromosome core.

SYCP3 localizes to the lateral elements of the synaptonemal complex and is essential for male meiosis. The genomic structure of SYCP3 consists of nine exons spanning approximately 14 kb. In mouse and rat, but not in hamster, the putative translation start of SYCP3 is present in the first exon. The putative promoter of SYCP3 was also cloned and shown to drive transcription of a reporter gene in somatic cells.

Synaptonemal complexes from DNase-treated rat pachytene chromosomes contain (GT)n and LINE/SINE sequences.

Purified chromosome cores (synaptonemal complexes) of rat pachytene chromosomes, from which the chromatin is removed by extensive DNase II digestion, retain a residual class of DNA, presumably the bases of chromatin loops. This synaptonemal complex-associated DNA, isolated by proteinase digestion and phenol extraction of purified DNase-treated synaptonemal complexes, and cloned in plasmid vector pEMBL18, has a length distribution of 50-500 bp. From a library of these fragments, 21 fragments were sequenced. Present in this sample are short 40-200-bp segments with greater than 80% identity to "long" and "short" interspersed repeated elements (LINE/SINEs), an excess of GT/CA tandem repeats and a number of unidentified sequences. The LINE/SINE segments may play a role in homology vs. nonhomology recognition during meiosis and the alternating purine-pyrimidine sequences have been implicated in genetic recombination. Their enrichment in synaptonemal complexes may be related to the synapsis and recombination functions of meiosis.

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization.

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell-carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell-carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E-selectin expression, which is mediated by eNOS production and p38 mitogen-activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel-induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.

Increase of intracellular pH in p53-dependent apoptosis of thymocytes induced by gamma radiation.

Irradiation with gamma rays induces apoptosis of thymocytes by a p53-dependent pathway, but its mechanism is not clear. In this study, we report that gamma-ray-induced apoptosis was associated with the intracellular alkalinization of the thymocytes. After exposure to gamma rays, thymocytes underwent apoptosis when cultured in vitro, and the degree of apoptosis was dependent on the incubation period: The longer the incubation period, the greater the number of cells undergoing apoptosis. However, this apoptosis could be inhibited by the acidic condition of the culture. There was a positive correlation between the pHi of thymocytes and the degree of apoptosis. Treatment with gamma radiation induced apoptosis as well as the elevation of the pHi in thymocytes. The intracellular pH was higher in pre-apoptotic thymocytes than in those that did not undergo apoptosis. Furthermore, apoptosis induced by gamma radiation was inhibited by cycloheximide, actinomycin D or the intracellular Ca2+ chelator, TMB-8. The p53 protein is induced after gamma irradiation. Thus it appears that intracellular pH is increased during the gamma-ray-induced p53-dependent apoptosis of thymocytes.

Infiltrated Cells in Experimental Allergic Encephalomyelitis by Additional Intracerebral Injection in Myelin-Basic-Protein-SensitizedB6 Mice.

We previously reported that murine experimental allergic encephalomyelitis can be induced by an additional intraperitoneal and intracerebral (i.c.) restimulation in resistant B6 mice after standard immunization with myelin antigens in complete Freund's adjuvant and Bordetella pertussis coadjuvant. Neutrophils infiltrated into perivascular spaces at 12 h, followed by mononuclear cells 24 h after i.c. injection. In this study, we report that the i.c. injection induced the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The kinetic expression of ICAM-1 or VCAM-1 on brain endothelial cells paralleled the infiltration of neutrophils and mononuclear cells, respectively. The infiltrated lymphocytes also expressed very late antigen-4 (VLA-4) molecules. The microvascular endothelial cells were positive for VCAM-1, whereas the surrounding mononuclear cells were VLA-4 positive. Furthermore, we found a unique subpopulation of cells with characteristics of CD4(-)CD8(-)V(beta)8(+) markers. The kinetic studies of this population showed that these cells were transiently depleted from 12 to 24 h after i.c. challenge (before the development of clinical symptoms) in cervical lymph nodes. These CD4(-)CD8(-)V(beta)8(+) cells can be expanded by in vitro culture with myelin basic protein or IL-2. No significant changes of CD4(+)/CD8(+) cells were noted. CD4(+)CD8(-)CD3(+) cells were also found in brain by double histochemical stains and were the major infiltrating cells at 24 or 48 h after i.c. challenge.

Tumour necrosis factor-alpha causes an increase in blood-brain barrier permeability during sepsis.

Blood-brain barrier (BBB) permeability during sepsis with Escherichia coli or Streptococcus pneumoniae was examined in a mouse model and measured by a circulating beta-galactosidase tracer. The leakage of brain microvascular vessels during sepsis was confirmed by transmission electron microscopic examination of brain tissues stained with horseradish peroxidase. The increase of BBB permeability induced by E. coli and S. pneumoniae, which was maximal at 3 h and 12 h after injection, respectively, was transient because of rapid clearance of the bacteria from the blood. Tumour necrosis factor-alpha (TNF-alpha) was stained on microvascular vessels of the brain during sepsis and intravenous injection of recombinant TNF-alpha also increased the BBB permeability. The increase in BBB permeability induced by either E. coli or S. pneumoniae could be inhibited by anti-TNF-alpha antibody. It was concluded that circulating TNF-alpha generated during sepsis induced the increase in BBB permeability.

Development of hematogenous pneumococcal meningitis in adult mice: the role of TNF-alpha.

Bacterial penetration across the blood-brain barrier (BBB) into the central nervous system is the first step in development of meningitis. The role of tumor necrosis factor-alpha (TNF-alpha) in the penetration process was examined with peripheral infection of Streptococcus pneumoniae type 6. After intraperitoneal infection of S. pneumoniae type 6, the BBB opening was increased continuously from 6 h and the mice died of septic shock within 36 h due to bacterial overgrowth. The bacteria crossed the BBB and began to deposit in brain at 6 h post infection. There was strong staining of TNF-alpha on blood vessels of brain from 6 h to 24 h post infection. Anti-TNF-alpha antibody blocked both the BBB opening and the entrance of circulatory S. pneumoniae type 6 into brain, indicating that TNF-alpha played an important role in controlling the opening of BBB. Furthermore, an adult murine model of hematogenous pneumococcal meningitis was developed that is based on opening of the BBB by TNF-alpha and controlling the degree of bacteremia by cefazolin antibiotic. In conclusion, hematogenous meningitis developed as TNF-alpha initiated BBB opening, peripheral bacteria entered into the brain and formed bacterial emboli, and then progressed to meningitis.

TNFalpha-induced cyclooxygenase 2 not only increases the vasopermeability of blood-brain barrier but also enhances the neutrophil survival in Escherichia coli-induced brain inflammation.

In Escherichia coli-induced brain inflammation, cyclooxygenase-2 was induced not only on brain arterioles at 3 h, but also on infiltrating neutrophils at 9 h post-intracerebral injection. Intravenous injection of E. coli or recombinant TNFalpha also induced cyclooxygenase-2 expression on arterioles. Cyclooxygenase-2 and TNFalpha were co-localized on the arterioles as well as the infiltrating neutrophils by serial-section staining, indicating that cyclooxygenase-2 was induced by TNFalpha. NS398 (a cyclooxygenase-2 selective inhibitor) not only inhibited the increase of blood-brain barrier permeability, but also enhanced the apoptosis of the infiltrating neutrophils after E. coli stimulation. This suggests that TNFalpha-stimulated cyclooxygenase-2 induction play an important role on E. coli-induced brain inflammation. Its inhibition would help the resolution of neutrophil-mediated brain inflammation.

Nutritional status of the water-soluble vitamins in an active Chinese elderly population in Hong Kong.

Dietary intakes of thiamine, riboflavin, nicotinic and ascorbic acid, together with the biochemical status of thiamine, riboflavin, pyridoxine and ascorbic acid, were determined in a cluster sample of 419 healthy active elderly subjects aged 60 years and above living in the community. Nicotinic acid intake per 1000 kcal (4.18 MJ) of food energy showed an age-related decrease in men, while women had higher ascorbic acid intakes than men. Between 38 and 98 per cent of this population have intakes of thiamine, riboflavin and nicotinic acid below the UK RDA values. Intakes of ascorbic acid were below the RDA for 17 per cent of men and 9 per cent of women. The prevalence of biochemical deficiency was 8, 14, 11.5 and 24 per cent for thiamine, riboflavin, pyridoxine and ascorbic acid respectively. A significant difference in intakes between groups with blood levels within and below the reference range was seen only for riboflavin, suggesting that factors other than low intake may be more important in contributing to low blood levels for thiamine and ascorbic acid. However, inaccuracies in dietary intake estimations may contribute to the poor correlation.

DNA binding proteins from Tetrahymena thermophila.

We have used DNA-cellulose chromatography to isolate single-strand binding proteins from Tetrahymena thermophila. Three major proteins which bind to denatured DNA-cellulose were obtained. The predominant protein has a molecular weight of 20 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis and possesses many of the properties of the helix destabilizing proteins isolated from prokaryotic and eukaryotic sources. The protein facilitates denaturation of the synthetic copolymer poly[d(A-T).d(A-T)], depressing the melting temperature by nearly 40 degrees C. It also permits the renaturation of poly[d(A-T)].d(A-T)] in high salt concentration. Two other binding proteins have molecular weight of 25 000 and 23 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The protein with a molecular weight of 25 000 is probably the "M protein" previously isolated from Tetrahymena thermophila which has been shown to stimulate Tetrahymena DNA polymerase. These two proteins failed to show helix destabilizing, DNA dependent ATPase, or deoxyribonuclease activities. These three proteins are abundant in the cell with approximately 1.0 x 10(6) to 10.0 x 10(6) molecules of each protein monomer per cell. One molecule of each protein monomer binds to 7 to 10 nucleotides as detected by a nitrocellulose filter binding assay. Peptide mapping of the three proteins suggests that they are all distinct. We have also found that the binding proteins can interact with Tetrahymena DNA polymerase and some other proteins to form an enzyme complex, a putative replication complex.

Activation of the Na(+)/H(+) antiporter, Na+/HCO3(-)/CO3(2-) cotransporter, or Cl(-)/HCO3(-) exchanger in spontaneous thymocyte apoptosis.

This study was undertaken to define the role of ion transporters on the apoptosis of thymocytes. Culture conditions, such as the ionic strength of NaCl, Ca2+, the buffer system (HCO3-/CO2), and the pH, could influence the spontaneous apoptosis of thymocytes. Depletion of NaCl in the culture medium either delayed or completely inhibited the apoptotic process of thymocytes, while its restoration led to a dose-dependent apoptosis. A high concentration (100 microM) of Ca2+ induced thymocyte apoptosis in the nominal absence of NaCl, whereas a low concentration (10 microM) enhanced apoptosis in the presence of 138 mM NaCl. Thymocytes had a higher spontaneous apoptotic rate in cultures without HCO3-/CO2 than in those with HCO3-/CO2. The thymocyte apoptosis completely ceased in medium at pH 6.0 and was considerably enhanced at pH 7.6. Intracellular pH, determined with the pH-sensitive bis-carboxyethyl carboxyfluorescein probe, was higher in apoptotic thymocytes than in nonapoptotic cells. Spontaneous apoptosis occurred in cells with alkaline intracellular condition, whereas it was considerably retarded in cells under acidified conditions. Amiloride analogue, including 5-(N,N-dimethyl)-amiloride (Na+/H+ antiporter), 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (Cl-/HCO3- exchanger), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (Na+/HCO3-/CO3(2-) cotransporter), inhibited the spontaneous thymocyte apoptosis. In contrast, neither cycloheximide nor actinomycin D had the same effect. In addition, thymocyte apoptosis was enhanced by PMA, but inhibited by forskolin. Taken together, thymocytes cultured in vitro underwent apoptosis with increased intracellular pH via activation of Na+/H+ antiporter, Na+/HCO3-/CO3(2-) cotransporter, or Cl-/HCO3- exchanger. This process does not require de novo protein synthesis.

Intracellular alkalinization in dexamethasone-induced thymocyte apoptosis.

Glucocorticoid can induce apoptosis of thymocytes, but its mechanism is not clear yet. In this study, we reported that dexamethasone-induced apoptosis was associated with intracellular alkalinization. Dexamethasone induced a higher percentage of apoptosis in 138 mM than in 50 mM NaCl, total abrogation of apoptosis was noted in NaCl-depleted culture medium. Highest apoptotic rate was observed in medium with pH 7.2, whereas it was partially and completely inhibited at pH 6.5 and pH 6.0, respectively. Intracellular pH was higher in pre-apoptotic thymocytes than non-apoptotic ones. The Na+/H+ antiporter inhibitor of 5-(N,N'-dimethyl)-amiloride inhibited the dexamethasone-induced increase in pHi and apoptosis of thymocytes. Glucocorticoid antagonist RU486 also blocked the dexamethasone-induced effect. Furthermore, the apoptosis and increase in intracellular pH induced by dexamethasone were inhibited by cycloheximide, actinomycin D. It seems that intracellular pH is increased during the development of thymocyte apoptosis and inhibiting its increment would retard the rate of progression to cell death.

A micronucleus-limited sequence family in Tetrahymena thermophila: organization and sequence conservation.

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronucleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited.

Evidence for intron capture: an unusual path for the evolution of proteins.

Most new genes are thought to evolve from preexisting genes but duplications of entire genes or shuffling of preexisting exons provides only a limited repertoire of new sequences that can be presented to a cell. Only pieces that previously existed can be used in the construction and any further divergence depends on the slow accumulation of mutations. We show here the presence of a small, in-frame intron in a ciliate phosphoglycerate kinase gene and the insertion of an unusually random amino acid sequence at the same position in trypanosome phosphoglycerate kinase. The unusual sequences in trypanosomes were likely to have originally been introns that have been subsequently captured by the protein and have now been incorporated as part of the coding sequence. Via this path a truly unique sequence can be incorporated into an existing protein, leading in time to the evolution of a new, functionally distinct protein.

Inhibition of Escherichia coli-induced meningitis by carboxyfullerence.

The effect of a water-soluble malonic acid derivative of carboxyfullerence (C60) against Escherichia coli-induced meningitis was tested. C60 can protect the mice from E. coli-induced death in a dose-dependent manner. C60 administered intraperitoneally as late as 9 h after E. coli injection was still protective. The C60-treated mice had less tumor necrosis factor alpha and interleukin-1beta production by staining of brain tissue compared to the levels of production for nontreated mice. The E. coli-induced increases in blood-brain barrier permeability and inflammatory neutrophilic infiltration were also inhibited. These data suggest that C60 is a potentially therapeutic agent for bacterial meningitis.

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase.

We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.

Protein coding gene trees in ciliates: comparison with rRNA-based phylogenies.

We have reexamined the phylogeny of the ciliates using alpha-tubulin and phosphoglycerate kinase gene sequences. For alpha-tubulin, we have compared the amino acid and nucleotide sequences of 20 species representing seven of the nine classes of the phylum (Karyorelictea, Heterotrichea, Hypotrichea, Oligohymenophorea, Colpodea, Nassophorea, and Litostomatea). The phylogenetic tree resembles a bush from which three monophyletic lineages can be distinguished which correspond to the three classes Hypotrichea, Oligohymenophorea, and Litostomatea. For phosphoglycerate kinase, we have compared the amino acid sequences from 7 species representing three classes (Heterotrichea, Hypotrichea, and Oligohymenophorea). The branching pattern is resolved in three deeply separated branches with an early emergence of the heterotrich. Our comparative analysis shows that if alpha-tubulin phylogeny is not informative at the interclass level, the preliminary data from the phosphoglycerate kinase molecule appear more promising. Nevertheless, at low taxonomic level and at the class level, the resolved phylogenetic relationships inferred from both protein and rRNA sequence data are congruent.

Effects of verapamil on coronary vascular resistance in rabbits: measurement with pulsed Doppler velocimetry.

BACKGROUND: Verapamil is an effective vasodilator. The purpose of this study was to investigate the in vivo effect of verapamil on coronary blood flow velocity and vascular resistance in anesthetized, open-chest rabbits. METHODS: Twenty-one male New Zealand white rabbits were anesthetized, and a 3-mm suction-type pulsed Doppler velocimeter probe was applied to the proximal part of the left anterior descending coronary artery after median sternotomy. The rabbits received intravenous bolus infusion of 4 different doses of verapamil (0.01 mg/kg, n = 5; 0.1 mg/kg, n = 5; 1 mg/kg, n = 5, and 10 mg/kg, n = 6). The percent changes in coronary blood flow velocity and coronary vascular resistance were examined. RESULTS: There was 10.0+/-1.6% increase in coronary blood flow (CBF) and 12.5+/-1.9% reduction in coronary vascular resistance (CVR) after infusion of 0.01 mg/kg of verapamil. The CBF increased 23.0+/-9.5% and CVR decreased 24.2+/-5.2% after infusion of 0.1 mg/kg of verapamil. Infusion of 1 mg/kg of verapamil induced 34.8+/-10.5% increase in CBF and 32.6+/-2.5% reduction in CVR. The CBF increased 41.1+/-14.8% and CVR decreased 45.1+/-5.4% after infusion of 10 mg/kg of verapamil. CONCLUSIONS: Compared with baseline condition, all doses of verapamil increased coronary blood flow velocity and decreased coronary vascular resistance significantly in anesthetized, open-chest rabbits.