Lactobacillus gasseri PA-3 directly incorporates purine mononucleotides and utilizes them for growth.

Lactococcus lactis has been reported unable to directly incorporate mononucleotides but instead requires their external dephosphorylation by nucleotidases to the corresponding nucleosides prior to their incorporation. Although Lactobacillus gasseri PA-3 (PA-3), a strain of lactic acid bacteria, has been found to incorporate purine mononucleotides such as adenosine 5'-monophosphate (AMP), it remains unclear whether these bacteria directly incorporate these mononucleotides or incorporate them after dephosphorylation to the corresponding nucleosides. This study evaluated whether PA-3 incorporated radioactively-labeled mononucleotides in the presence or absence of the 5'-nucleotidase inhibitor α,ß-methylene ADP (APCP). PA-3 took up 14C-AMP in the presence of APCP, as well as incorporating 32P-AMP. Furthermore, radioactivity was detected in the RNA/DNA of bacterial cells cultured in the presence of 32P-AMP. Taken together, these findings indicated that PA-3 incorporated purine mononucleotides directly rather than after their dephosphorylation to purine nucleosides and that PA-3 utilizes these purine mononucleotides in the synthesis of RNA and DNA. Although additional studies are required to identify purine mononucleotide transporters in PA-3, this study is the first to show that some lactic acid bacteria directly incorporate purine mononucleotides and use them for growth.

Species-dependent patterns of incorporation of purine mononucleotides and nucleosides by lactic acid bacteria.

Although most lactic acid bacteria do not directly incorporate purine nucleotides, the strain Lactobacillus gasseri PA-3 was found to incorporate purine mononucleotides. To determine whether the direct uptake of purine mononucleotides is dependent on the species or strain of lactic acid bacteria, incorporation of purine mononucleotides was assessed in L. gasseri, Lactcoccus lactis sbsp. lactis, Streptococcus thermophilus and other species of lactic acid bacteria. Each bacterial strain was incubated with 32P-AMP or 14C-adenosine and the incorporation of each purine was evaluated by measuring their radioactivity. All investigated strains of L. gasseri incorporated 32P-AMP, whereas strains of S. thermophilus and most strains of L. lactis did not. Incorporation of 32P-AMP into strains of Pediococcus was dependent on the strain or species of that genus of bacteria. All investigated strains, except for one strain of L. gasseri, incorporated 14C-adenosine, with S. thermophilus, L. lactis and Pediococcus generally displaying greater incorporation of 14C-adenosine than L. gasseri. Although most lactic acid bacteria such as S. thermophiles and L. lactis do not incorporate purine mononucleotides, some species such as L. gasseri directly incorporate purine mononucleotides. These findings indicate that the preferential incorporation of purine mononucleotides or nucleosides by lactic acid bacteria is dependent on the species or strain.

Lactobacillus gasseri PA-3, but not L. gasseri OLL2996, reduces the absorption of purine nucleosides in rats.

Lactobacillus gasseri PA-3 (PA-3) is a bacterial strain with a strong ability to degrade purine nucleosides. We previously showed that PA-3 incorporates purines in vitro and that oral administration of PA-3 and purines to rats attenuated their absorption of purines. It remains unclear whether these effects of PA-3 depend on bacterial strains. This study therefore compared the abilities of PA-3 and another bacterial strain of L. gasseri, OLL2996, which has shown decreased ability to degrade purine nucleosides in vitro, to incorporate purine nucleosides and to inhibit the absorption of purines fed to rats. Each bacterial strain was incubated in the presence of 14C-adenosine or 14C-inosine and the incorporation of each purine was evaluated by measuring their radioactivity. In vivo, rats were fed 14C-labeled purines along with PA-3 or OLL2996 and the absorption of these 14C-labeled purines was evaluated by analyzing radioactivity of blood samples. PA-3 incorporated about twice as much 14C-adenosine and 14C-inosine as OLL2996. The elevation of radioactivity levels in blood was 10-20% lower in rats treated with PA-3 than in control rats, after feeding with both 14C-adenosine and 14C-inosine as purines. In contrast, treatment with OLL2996 did not have statistically significant effects on radioactivity compared with the control group. These results indicate that the magnitude of bacterial inhibition of purine absorption is dependent on bacterial strain, correlating at least partly with the ability to incorporate and degrade purines.

Lactobacillus gasseri PA-3 utilizes the purines GMP and guanosine and decreases their absorption in rats.

Excessive intake of purine-rich foods elevates serum uric acid levels, making it a risk factor for hyperuricemia. We hypothesized that lactic acid bacteria ingested with food might utilize purines and contribute to their decreased absorption in the intestines, thereby preventing hyperuricemia. We previously reported that Lactobacillus gasseri PA-3 (PA-3) incorporates adenosine/inosine and related purines and that oral ingestion of PA-3 reduced the absorption of these purines in rats. However, it is unclear whether PA-3 also decreases the absorption of other purines, such as guanosine 5'-monophosphate (GMP) and guanosine. This study investigated whether PA-3 incorporates GMP and guanosine and reduces their absorption in rats. PA-3 incorporated both purines, with 14C-GMP uptake being greater than that of 14C-guanosine. Radioactivity in rat blood was significantly lower 30, 45, and 60 minutes after administration of 14C-GMP plus PA-3 than after administration of 14C-GMP alone and was significantly lower 15 minutes after administration of 14C-guanosine plus PA-3 than after administration of 14C-guanosine alone. PA-3 incorporates GMP and guanosine in vitro. Oral administration of PA-3 with GMP and guanosine reduces the intestinal absorption of these purines in vivo. These findings, together with those of previous studies, indicate that PA-3 reduces the absorption of major purines contained in foods. PA-3 may also attenuate the excessive absorption of dietary purines in humans, protecting these individuals against hyperuricemia.

Oral administration of a medium containing both D-aspartate-producing live bacteria and D-aspartate reduces rectal temperature in chicks.

1. The aim of this study was to investigate the effects on the rectal temperature of young chicks of the oral administration of a medium that contained both live bacteria that produce D-aspartate (D-Asp) and D-Asp. 2. In Experiment 1, chicks were subjected to chronic oral administration of either the medium (containing live bacteria and 2.46 µmol D-Asp) or water from 7 to 14 d of age. Plasma-free amino acids as well as mitochondrial biogenic gene expression in the breast muscle were analysed. In Experiment 2, 7-d-old chicks were subjected to acute oral administration of the above medium or of an equimolar amount of D-Asp to examine their effect on changes in rectal temperature. In Experiment 3, after 1 week of chronic oral administration of the medium, 14-d-old chicks were exposed to either high ambient temperature (HT; 40 ± 1°C, 3 h) or control thermoneutral temperature (CT; 30 ± 1°C, 3 h) to monitor the changes in rectal temperature. 3. Chronic, but not acute, oral administration of the medium significantly reduced rectal temperature in chicks, and a chronic effect also appeared under HT conditions. 4. Chronic oral administration of the medium significantly reduced the mRNA abundance of the avian uncoupling protein (avUCP) in the breast muscle, but led to a significant increase in avian adenine nucleotide translocator (avANT) mRNA in the same muscle. 5. (a) These results indicate that the medium can reduce body temperature through the decline in avUCP mRNA expression in the breast muscle that may be involved in reduced mitochondrial proton leaks and heat production. (b) The increase in avANT further suggests a possible enhancement of adenosine triphosphate (ATP) synthesis.

Effects of Lactobacillus plantarum Strain OLL2712 Culture Conditions on the Anti-inflammatory Activities for Murine Immune Cells and Obese and Type 2 Diabetic Mice.

Studies on the health-promoting effects of lactic acid bacteria (LAB) are numerous, but few provide examples of the relationship between LAB function and culture conditions. We verified the effect of differences in culture conditions on Lactobacillus plantarum OLL2712 functionality; this strain exhibits anti-inflammatory activity and preventive effects against metabolic disorders. We measured interleukin-10 (IL-10) and IL-12 production in murine immune cells treated with OLL2712 cells prepared under various culture conditions. The results showed that the IL-10-inducing activities of OLL2712 cells on murine immune cells differed dramatically between OLL2712 groups at different culture phases and using different culture medium components, temperatures, and neutralizing pHs. In particular, exponential-phase cells had much more IL-10-inducing activity than stationary-phase cells. We confirmed that the Toll-like receptor 2 (TLR2) stimulation activity of OLL2712 cells depended on culture conditions in conjunction with IL-10-inducing activity. We also demonstrated functional differences by culture phases in vivo; OLL2712 cells at exponential phase had more anti-inflammatory activity and anti-metabolic-disorder effects on obese and diabetic mice than those by their stationary-phase counterparts. These results suggest that culture conditions affect the functionality of anti-inflammatory LAB.IMPORTANCE While previous studies demonstrated that culture conditions affected the immunomodulatory properties of lactic acid bacteria (LAB), few have comprehensively investigated the relationship between culture conditions and LAB functionality. In this study, we demonstrated several culture conditions of Lactobacillus plantarum OLL2712 for higher anti-inflammatory activity. We also showed that culture conditions concretely influenced the health-promoting functions of OLL2712 in vivo, particularly against metabolic disorders. Further, we characterized a novel mechanism by which changing LAB culture conditions affected immunomodulatory properties. Our results suggest that culture condition optimization is important for the production of LAB with anti-inflammatory activity.

Evaluation of purine utilization by Lactobacillus gasseri strains with potential to decrease the absorption of food-derived purines in the human intestine.

It is well accepted that frequent and heavy intake of purine-rich foods causes elevation of serum uric acid levels, which is a risk factor of hyperuricemia. Reducing intestinal absorption of dietary purines may attenuate the elevation of serum uric acid levels and exacerbation of hyperuricemia. This reduction may be achieved by the ingestion of lactic acid bacteria that take up purines in the intestine. In this study, we investigated the degree of uptake and utilization of purines of three lactobacilli strains. Among them, Lactobacillus gasseri PA-3 (PA-3) showed the greatest incorporation of 14C-adenine. PA-3 also incorporated 14C-adenosine and 14C-AMP. Additionally, using defined growth medium, PA-3 demonstrated greater proliferation in the presence of these purines than in their absence. Although further investigation is required, ingestion of PA-3 may lower serum uric acid levels by reducing intestinal absorption of purines in humans.

Objective tumor response to denosumab in patients with giant cell tumor of bone: a multicenter phase II trial.

BACKGROUND: Giant cell tumor of bone (GCTB) is a rare primary bone tumor, characterized by osteoclast-like giant cells that express receptor activator of nuclear factor-kappa B (RANK), and stromal cells that express RANK ligand (RANKL), a key mediator of osteoclast activation. A RANKL-specific inhibitor, denosumab, was predicted to reduce osteolysis and control disease progression in patients with GCTB. PATIENTS AND METHODS: Seventeen patients with GCTB were enrolled. Patients were treated with denosumab at 120 mg every 4 weeks, with a loading dose of 120 mg on days 8 and 15. To evaluate efficacy, objective tumor response was evaluated prospectively by an independent imaging facility on the basis of prespecified criteria. RESULTS: The proportion of patients with an objective tumor response was 88% based on best response using any tumor response criteria. The proportion of patients with an objective tumor response using individual response criteria was 35% based on the modified Response Evaluation Criteria in Solid Tumors (RECIST) criteria, 82% based on the modified European Organization for Research and Treatment of Cancer (EORTC) criteria, and 71% based on inverse Choi criteria. The median time of study treatment was 13.1 months. CONCLUSION: The findings demonstrate that denosumab has robust clinical efficacy in the treatment of GCTB.

Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro.

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.

Production of recombinant human bile salt-stimulated lipase in Pichia pastoris.

Recombinant human bile salt-stimulated lipase (rhBSSL) was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris. Human BSSL has 16 successively repeated sequences in the carboxy terminal region. The sequence consists of 11 amino acid residues. The coding sequence for the middle 11 of the 16 repeats was removed from hBSSL cDNA to facilitate efficient secretory expression. The clone used for fermentation was a transformant of GS115 (his4) integrated with four copies of the expression cassette containing the modified hBSSL cDNA. Unique fermentation conditions were required for efficient expressions of rhBSSL in the high cell-density fermentation. A sufficient glycerol feed at 30 degrees C and pH 4 under an adequate concentration of dissolved oxygen in the growth phase make the cells active over a long induction period of approximately 15 days. On methanol induction, the concentration of dissolved oxygen should be maintained very low in the presence of sorbitol and skimmed milk at 20 degrees C and pH 5.7. Under these conditions, 0.8-1 g of rhBSSL was secreted in 1 liter of the medium. By immunoelectron microscopy, rhBSSL-tagged gold particles were located in secretion microbodies after the beginning of methanol induction. The secreted rhBSSL was efficiently captured and purified by expanded bed adsorption chromatography.

Role of DNA ligase in the illegitimate recombination that generates lambdabio-transducing phages in Escherichia coli.

We studied the role of DNA ligase in illegitimate recombination in Escherichia coli. A temperature-sensitive mutation in the lig gene reduced the frequency with which lambdabio-transducing phages were generated to 10-14% of that of wild type under UV irradiation. Reintroduction of the lig gene into this mutant restored the frequency of recombinant phage generation to that of wild type. Furthermore, overexpression of DNA ligase enhanced illegitimate recombination by 10-fold with or without UV irradiation. In addition, when DNA ligase was present in only limited amounts, UV-induced or spontaneous illegitimate recombination occurred exclusively at hotspot sites that have relatively long sequences of homology (9 or 13 bp). However, when DNA ligase was overexpressed, most of the illegitimate recombination took place at non-hotspot sites having only short sequences of homology (<4 bp). Thus, the level of ligase activity affects the frequency of illegitimate recombination, the length of sequence homology at the recombination sites, and the preference for recombination at hotspots, at least after UV irradiation. These observations support our hypothesis that the illegitimate recombination that generates lambdabio-transducing phages is mediated by the DNA break-and-join mechanism.

An approach to the removal of yeast specific O-linked oligo-mannoses from human midkine expressed in Pichia pastoris using site-specific mutagenesis.

Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal. The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine. However, more than half of the product receives yeast specific mannosylations. The sites for the mannosylations were determined to be the three threonine residues in the carboxy-terminal region of human midkine. In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis. HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues. Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

Control of genetic stability in Escherichia coli: the SbcB 3'-5' exonuclease suppresses illegitimate recombination promoted by the RecE 5'-3' exonuclease.

BACKGROUND: The Escherichia coli sbcB gene, which codes for a 3'-5' exonuclease, ExoI, is known to suppress illegitimate recombination. In contrast, the recE gene, which codes for a 5'-3' exonuclease, Exo VIII promotes joining between DNA ends having short stretches of homology. Therefore, it seems likely that the 3'-5' and 5'-3' exonucleases regulate genetic instability that is mediated by illegitimate recombination. However, there has been little evidence to substantiate the involvement of exonuclease activity in the promotion and suppression of illegitimate recombination. RESULTS: Using a plasmid system for the analysis of deletion formation, we first demonstrated that deletion formation is increased by the sbcA mutation, which activates the expression of RecE 5'-3' exonuclease. It is thought that DNA ends having 3'-single stranded overhangs are important for illegitimate recombination. Next, we found that a large supply of SbcB 3'-5' exonuclease suppresses the deletion formation enhanced by the RecE exonuclease. Moreover, the SbcB exonuclease even suppressed deletion formation in cells not expressing RecE exonuclease. CONCLUSION: We conclude that DNA ends with 3'-overhangs produced by 5'-3' dsDNA exonuclease activity are proficient for illegitimate recombination, while blunt DNA ends produced by 3'-5' ssDNA exonuclease activity are deficient for illegitimate recombination. Therefore, both exonucleases may play important roles in genetic stability by controlling end-joining between DNA molecules.

Value of acceleration flow and the prestenotic to stenotic coronary flow velocity ratio by transthoracic color Doppler echocardiography in noninvasive diagnosis of restenosis after percutaneous transluminal coronary angioplasty.

OBJECTIVES: The study evaluated the value of coronary flow velocity measurement by transthoracic color Doppler echocardiography (TTCDE) for the noninvasive diagnosis of restenosis after percutaneous transluminal coronary angioplasty (PTCA) for left anterior descending coronary artery (LAD) lesions. BACKGROUND: Recent advances in TTCDE provide coronary flow velocity measurements in the LAD under the guidance of color flow mapping. METHODS: We studied 53 patients who underwent successful PTCA for LAD lesions and follow-up coronary angiography (18 patients with restenosis [Group-R], 35 patients without restenosis [Group-N]). We searched localized color aliasing corresponding to local flow acceleration to obtain coronary flow velocity at PTCA sites in the LAD. When localized aliasing was detected, we measured coronary flow velocity at the aliasing (stenotic site) and the prestenotic site. RESULTS: Using TTCDE, it was possible to measure mean diastolic velocity (MDV) in the LAD in 41 (77%) of 53 patients (14 of 18 patients in Group-R; 27 of 35 patients in Group-N). Localized aliasing was displayed by color flow mapping in 14 (100%) of 14 patients in Group-R, and 15 (56%) of 27 patients in Group-N. Stenotic MDV in Group-R was significantly higher than that in Group-N (60.3 +/- 21.1 vs. 35.1 +/- 7.6 cm/s, p < 0.01), although prestenotic MDV did not differ between Group-R and Group-N (20.2 +/- 3.0 vs. 19.6 +/- 2.3 cm/s). There were significant differences in the prestenotic to stenotic MDV ratio between Group-R and Group-N (0.36 +/- 0.10 vs. 0.57 +/- 0.09, p < 0.001). Localized aliasing with the prestenotic to stenotic MDV ratio <0.45 as the optimal cutoff value had a sensitivity of 86% and a specificity of 93% for the presence of restenosis in LAD lesions. CONCLUSIONS: Detection of localized color aliasing and measurement of the prestenotic to stenotic MDV ratio in the LAD by TTCDE are useful in the noninvasive diagnosis of restenosis after PTCA for LAD lesions.

Production of recombinant human midkine in yeast, Pichia pastoris.

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.

Inhibitory effect of TS-962 on the formation of early atherosclerotic lesions in high fat-fed hyperlipidemic hamsters.

The hypocholesterolemic and anti-atherosclerotic effect of TS-962, a specific ACAT inhibitor, was investigated in a hamster model fed a high fat diet containing 10% coconut oil and 0.05% cholesterol. Lipid accumulated atherosclerotic lesions were detected by using oil red O staining in the lesion-prone aortic arch. A high dose, estimated to be 15 mg/kg, of TS-962 decreased serum cholesterol to normal levels with reduction of liver cholesterol contents below normal levels, and as a consequence, entirely inhibited the lipid accumulation in the aortic arch. Furthermore, a low dose, estimated to be 1.5 mg/kg, of TS-962 remarkably inhibited aortic lipid accumulation by 73% compared with the control group, without changing either serum cholesterol level or liver cholesterol content. These findings suggest that TS-962 is effective in the primary prevention of atherosclerosis by directly suppressing the formation of foam cells in arteries.

Up-regulation of low density lipoprotein receptor by a novel isobenzofranone derivative, MD-700.

Stimulatory effects of a novel isobenzofranone, MD-700, on low density lipoprotein (LDL) receptor activity were investigated in vitro and in vivo. MD-700 at 0.03 microg/ml elevated the expression of LDL receptor in HepG2 cells within 4 h. Corresponding to this, uptake of fluorescent labeled-LDL (3,3'-dioctadecylindocarbocyanine-LDL) by the cells increased linearly in time- and dose-dependent manner by MD-700 for up to 12 h. In the experiment using HepG2 cells transiently transfected with promoter-luciferase gene constructs, MD-700 increased luciferase activity in a dose-dependent manner from 0.03 to 0.1 microg/ml. In contrast, luciferase activity was not stimulated by MD-700 in construct with a deleted sterol regulatory element (SRE)-1, suggesting importance of SRE-1 in stimulation of the LDL receptor gene promoter by MD-700. Binding experiments on liver membranes from MD-700-treated hamsters showed about a 60% increase in 125I-labeled LDL binding. A Scatchard plot revealed that MD-700 increased the maximal binding without affecting binding affinity. In contrast to findings with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin, MD-700 had no effect on the sterol synthesis in hamster liver homogenates. These results suggest that MD-700 stimulates the expression of LDL receptor, presumably in a manner independent of change in sterol metabolism, and thereby promotes LDL clearance. Hypocholesterolemic actions of MD-700 in hamsters were then examined. MD-700 lowered serum cholesterol levels in hamsters fed normal chow or a high-fat diet. Fractionation of serum lipoproteins demonstrated that MD-700 selectively decreased LDL and very low density lipoprotein cholesterol. Dose-dependent decrease in serum cholesterol was also seen in hypercholesterolemic rats. Thus, the hypocholesterolemic action of MD-700 may be attributed to up-regulation of the LDL receptor, based on stimulation of the transcription of the LDL receptor gene. Although pravastatin stimulates LDL uptake and lowers serum cholesterol in a manner similar to that seen with MD-700, the mechanism responsible for hypocholesterolemic action appears to differ.

[Assessment of coronary flow reserve in patients with hypertrophic cardiomyopathy using transthoracic color Doppler echocardiography].

Abnormal coronary flow pattern and coronary vasodilator reserve have been identified in patients with hypertrophic cardiomyopathy (HCM) using invasive techniques. The characteristics of coronary flow velocity and coronary flow reserve were evaluated by noninvasive-recording of coronary flow velocity in the distal portion of the left anterior descending coronary artery in 7 patients with HCM and 7 normal subjects using transthoracic color Doppler echocardiography. Coronary flow velocity was measured at rest and during intravenous infusion of adenosine triphosphate (0.15 mg/kg/min). Diastolic peak velocity, diastolic mean velocity, the time from the beginning of diastole to peak velocity (TVP) and velocity half time from peak velocity was measured in each group. Coronary flow reserve was obtained as the ratio of hyperemic mean velocity to resting mean velocity. TVP was significantly prolonged in the patients with HCM compared with the normal subjects (159 +/- 38 vs 103 +/- 54 msec, p < 0.05). Velocity half time was significantly shorter in the patients with HCM compared with the normal subjects (304 +/- 138 vs 451 +/- 109 msec, p < 0.05). Although diastolic mean velocity during hyperemia was not different between the 2 groups (62 +/- 8 vs 70 +/- 19 cm/sec), diastolic mean velocity at rest was significantly higher in the patients with HCM than in the normal subjects (39 +/- 6 vs 26 +/- 7 cm/sec, p < 0.01). Therefore, coronary flow reserve was significantly lower in the patients with HCM than in the normal subjects (1.6 +/- 0.4 vs 2.7 +/- 0.4, p < 0.001). There was a good correlation between diastolic mean velocity and the ratio of interventricular septal to posterior left ventricular wall thickness (y = 0.024x + 0.46, r = 0.75). Transthoracic assessment of coronary flow velocity using color Doppler echocardiography reveals that coronary flow reserve is reduced in patients with HCM because of increased baseline resting diastolic mean velocity.