Warfarin as a therapeutic option in the control of chronic cluster headache: a report of three cases.

Chronic cluster headache remains refractory to medical therapy in at least 30% of those who suffer from this condition. The lack of alternative medical therapies that are as effective as, or more effective than, lithium carbonate makes new therapies necessary for this highly disabling condition. Based on a previous report, we gave oral anticoagulants to three patients with chronic cluster headache. Two of them remained cluster headache-free while taking warfarin. In the third patient, the use of warfarin for three weeks initially increased the frequency and intensity of cluster headache attacks but subsequently induced a prolonged remission. In spite of the paucity of data available, oral anticoagulation appears to be a promising therapy for chronic cluster headache.

Production of menaquinones by lactic acid bacteria.

Lactic acid bacteria were examined for their ability to produce quinone compounds, which may include dietary sources of menaquinones. Isoprenyl quinones in bacterial cells grown in a synthetic medium were extracted and analyzed by thin layer chromatography. Lactococcus lactis ssp. cremoris (three strains), Lactococcus lactis ssp. lactis (two strains), and Leuconostoc lactis were selected as high producers of quinone that synthesized more than 230 nmol of quinones/g of dried cells. The quinones were presumed to be menaquinone-7 to -10 by high performance liquid chromatography. Precise molecular weights were determined by mass spectrometry for Lactococcus lactis ssp. cremoris YIT 2011 and Leuconostoc lactis YIT 3001 and identified as menaquinone-8 and -9 for the former and menaquinone-9 and -10 for the latter. Those strains, when grown either in reconstituted nonfat dry milk or a soymilk medium, produced a beneficial quantity for dietary supplement (i.e., 29 to 123 micrograms of menaquinones/L of the fermented medium).

Intestinal flora is not an intermediate in the phylloquinone-menaquinone-4 conversion in the rat.

To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinone-4 (MK-4) we investigated the tissue distribution of vitamin K in germ-free rats. The rats were made vitamin K deficient by feeding a vitamin K-free diet for 13 days. In a subsequent period of 6 days, phylloquinone and menadione were supplied via the drinking water in concentrations of 10 and 50 micromol l(-1). Menadione supplementation led to high levels of tissue MK-4, particularly in extrahepatic tissues like pancreas, aorta, fat and brain. Liver and serum were low in MK-4. Phylloquinone supplementation resulted in higher phylloquinone levels in all tissues when compared with vitamin K-deficient values. The main target organs were liver, heart and fat. Remarkably, tissue MK-4 levels were also higher after the phylloquinone supplementation. The MK-4 tissue distribution pattern after phylloquinone intake was comparable with that found after menadione intake. Our results demonstrate that the conversion of phylloquinone into MK-4 in extrahepatic tissues may occur in the absence of an intestinal bacterial population and is tissue specific. A specific function for extrahepatic MK-4 or a reason for this biochemical conversion of phylloquinone into MK-4 remains unclear thus far.

Transcriptional regulation of the Bacillus subtilis menp1 promoter.

The Bacillus subtilis men genes encode biosynthetic enzymes for formation of the respiratory chain component menaquinone. The menp1 promoter previously was shown to be the primary cis element for menFD gene expression. In the present work, it was found that either supplementation with nonfermentable carbon sources or reutilization of glycolytic end products increased menp1 activity in the late postexponential phase. The effect on menp1 activity by a particular end product (such as acetoin or acetate) was prevented by blocking the corresponding pathway for end product utilization. Alteration of a TGAAA motif within the promoter region resulted in unregulated menp1 activity throughout the culture cycle, irrespective of the carbon source added.

Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus.

Small-colony variants (SCVs) of Staphylococcus aureus were cultured from five patients with persistent and relapsing infections. All five SCV strains were nonhemolytic and nonpigmented and grew very slowly on routine culture media in an ambient atmosphere. In several instances, these phenotypic characteristics led to the initial misidentification of the organisms in the clinical microbiology laboratory. All four strains available for further analysis were shown to be auxotrophs that reverted to normal growth and morphology in the presence of menadione, hemin, and/or a CO2 supplement. Similarly, these isolates were resistant to aminoglycosides under routine conditions but susceptible in the presence of the metabolic supplements. For two patients, the large and small colony forms isolated concurrently were indistinguishable when analyzed by pulsed field gel electrophoresis and thus represented phenotypic variants within individual clones. We propose a model relating the phenotypic characteristics of S. aureus SCVs with the clinical pattern of persistent and relapsing infection.

Reduction of vitamin K2 concentrations in human liver associated with the use of broad spectrum antimicrobials.

It is unclear whether menaquinones produced by the intestinal microflora play any role in human nutrition. Reports of coagulopathy due to vitamin K deficiency occurring in patients receiving broad spectrum antibiotics indirectly suggest that vitamin K2 produced by the gut microflora may be utilized by the host. We analyzed the vitamin K1 (phylloquinone) and vitamin K2 (menaquinone) content in a convenience sample of 22 human post-mortem liver samples, including 9 individuals who had been receiving broad spectrum antimicrobials prior to death and 13 individuals who had been victims of sudden, unexpected deaths. There were no significant differences in the mean (+/- SEM) phylloquinone content between the 2 groups [21.9 (+/- 15.5) vs. 16.0 (+/- 9.3) pmol/g wet weight (excluding those who had received supplemental vitamin K1)] but there was a significant difference (p < 0.05) in the total menaquinone (MK) content, 70.0 (+/- 23.3) vs. 423.1 (+/- 141) pmol/g between the 2 groups. These findings suggest an association between receipt of broad spectrum antibiotics and a reduction in hepatic menaquinone concentration, lending support to the hypothesis that a reduction in the gut microflora responsible for their production leads to reduced hepatic stores of this form of the vitamin.

Dietary modification of potential vitamin K supply from enteric bacterial menaquinones in rats.

Rats given a low-fibre diet based on boiled white rice developed symptoms of severe vitamin K deficiency within 23 d. Inclusion of autoclaved black-eye beans (Vigna unguiculata) in the diet prevented the bleeding syndrome. To test the hypothesis that deficiency resulted from low phylloquinone intake exacerbated by inadequate production of menaquinones by the enteric bacteria, a follow-up experiment was carried out in which groups of rats were given an all-rice diet, a rice + beans diet or a stock diet. Rats on the all-rice diet had significantly lower faecal concentrations of the main menaquinone-producing bacterial species (Bacteroides fragilis and Bacteroides vulgatus) than animals on either of the other two diets. This coupled with the much lower faecal output on this diet suggests that total menaquinone production was low for the all-rice diet. The alterations in faecal flora were associated with several significant changes in caecal metabolism. Rats given the stock diet had much shorter caecal transit times and a considerably greater proportion of butyric acid in volatile fatty acid end-products than did rats on either of the other two diets.

Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli.

Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor. A strain so obtained (E. coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid. This shows that the gene (entC) encoding isochorismate synthase was mutated. E. coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin. When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis. The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene. The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster. The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis. We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.

Menaquinone biosynthesis in Bacillus subtilis: isolation of men mutants and evidence for clustering of men genes.

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis were selected by simultaneous resistance to two aminoglycoside antibiotics. These men mutants fell into two groups: group I, in which the nutritional requirement was satisfied either by o-succinylbenzoic acid or by 1,4-dihydroxy-2-naphthoic acid; and group II, comprising those capable of growing only when supplemented with 1,4-dihydroxy-2-naphthoic acid. The latter group could be further subdivided into two classes on the basis of syntrophy experiments, fine-structure genetic mapping, and in vitro complementation by cell-free extracts (Meganathan et al., J. Bacteriol., 145:328-332, 1981). These subclasses of group II defined the menB and menE genes, whereas group I appeared to comprise mutations in the menC and menD genes. All of the men mutations tested, whether occurring in menB, menE, or menC,D, could be placed by genetic mapping with bacteriophage PBS1 between bioB and ald on the B. subtilis genome.

Role of vitamin K2 in the organization and function of Staphylococcus aureua membranes.

A mutant of Staphylococcus aureus auxotrophic for menadione (a vitamin K2 precursor) was used to study the effects of menadione deprivation on the structure and function of the cell membrane. The phospholipid composition and metabolism was essentially unaltered by menadione deprivation. Removal of this percursor caused cellular levels of the cytochromes, protoheme, vitamin K2, and several membrane-bound flavoprotein dehydrogenase activities to decrease as a function of growth dilution. The cytochromes were enzymatically reducible and maintained in the same proportions as menadione-supplemented cells. Oxidative phosphorylation, however, was reduced more than 10-fold and membrane vesicles obtained from menadione-deprived cells were unable to couple glycine transport to L-lactate oxidation. Succinic dehydrogenase and adenosine 5' triphosphate hydrolysis appeared unaffected by menadione deprivation. These data suggest that menadione deprivation in the mutant stops the synthesis of vitamin K2 and other electron transport chain components and prosthetic groups. Although individual electron transport chain members remained fully functional during menadione deprivation, the overall efficiency of the chain, measured in terms of its function in electron transport, oxidative phosphorylation, and electron transport chain-linked transport, dropped greatly. This suggests that the synthesis of vitamin K2 is modulated to the synthesis of other components of the electron transport system, and that their organization into a functional system requires a specific concentration of vitamin K2 with respect to total membrane lipid.