Coffee consumption and mortality in Japan with 18 years of follow-up: the Jichi Medical School Cohort Study.

OBJECTIVE: Coffee consumption can be expected to reduce mortality due to cardiovascular diseases and cancer. This study tested the hypothesis of an inverse association between coffee intake and all-cause mortality and mortality due to cancer, coronary heart disease, or stroke. STUDY DESIGN: Prospective cohort study. METHODS: We analyzed data from the Jichi Medical School Cohort Study, Japan, enrolling 9946 subjects (men/women: 3870/6,076, age: 19-93 years) from 12 communities. A food frequency questionnaire assessing the subjects' daily coffee consumption was used. RESULTS: During an average follow-up of 18.4 years, the total number of deaths was 2024, including 677 for cancer, 238 for coronary heart disease, and 244 for stroke. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause mortality and cause-specific mortality due to cancer, coronary heart disease, and stroke. Overall, no significant association was shown between coffee consumption and all-cause mortality. In the cause-specific mortality analyses, stroke mortality was significantly lower in those who consumed 1-2 cups of coffee daily (HR [95% CI]: 0.63 [0.42-0.95]) than in those who do not consume coffee, and this association occurred only in men. CONCLUSION: This study showed no significant association between coffee consumption and all-cause mortality. A U-shaped association between coffee consumption and stroke mortality with a 37% lower stroke mortality, only significant in men who consume 1-2 cups of coffee daily was observed. It is necessary to examine the possibility of intervention studies to reduce stroke mortality through coffee consumption.

Factors secreted from dental pulp stem cells show multifaceted benefits for treating experimental temporomandibular joint osteoarthritis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by progressive cartilage degeneration, abnormal bone remodeling, and chronic pain. In this study, we aimed to investigate effective therapies to reverse or suppress TMJOA progression. DESIGN: To this end, we performed intravenous administration of serum free conditioned media from human exfoliated deciduous teeth stem cells (SHED-CM) into a mechanical-stress induced murine TMJOA model. RESULTS: SHED-CM administration markedly suppressed temporal muscle inflammation, and improved bone integrity and surface smoothness of the destroyed condylar cartilage. Moreover, SHED-CM treatment decreased the number of IL-1ß, iNOS, and MMP-13 expressing chondrocytes, whereas it specifically increased PCNA-positive cells in the multipotent polymorphic cell layer. Notably, the numbers of TdT-mediated dUTP nick end labeling (TUNEL)-positive apoptotic chondrocytes in the SHED-CM treated condyles were significantly lower than in those treated with DMEM, whereas the proteoglycan positive area was restored to a level similar to that of the sham treated group, demonstrating that SHED-CM treatment regenerated the mechanical-stress injured condylar cartilage and subchondral bone. Secretome analysis revealed that SHED-CM contained multiple therapeutic factors that act in osteochondral regeneration. CONCLUSIONS: Our data demonstrated that SHED-CM treatment promoted the regeneration and repair of mechanical-stress induced mouse TMJOA. Our observations suggest that SHED-CM has potential to be a potent tissue-regenerating therapeutic agent for patients with severe TMJOA.

Elevated arterial stiffness in postmenopausal women with osteoporosis.

OBJECTIVES: Osteoporosis and increased pulse wave velocity (PWV) are cardiovascular risk factors. We investigated the relationship between PWV and bone mass in the lumbar spine in postmenopausal women. METHODS: We studied the PWV in 95 women; 38 postmenopausal women with normal spinal bone mineral density (BMD), 32 osteopenic postmenopausal women, and 25 osteoporotic postmenopausal women. The brachial-ankle PWV (baPWV) was measured using an automated device. The BMD of the lumbar spine (L2-L4) was measured using dual-energy X-ray absorptiometry. RESULTS: After adjusting for age and years since menopause, women with osteoporosis had a significantly higher baPWV than those with normal BMD (1500 +/- 220 cm/s versus 1340 +/- 215 cm/s; P < 0.05), but no significant differences in baPWV were seen between the osteoporotic and osteopenic groups or between the osteopenic and normal BMD groups. In univariate regression analysis, the baPWV was significantly negatively correlated with BMD (r = -0.450, P < 0.01), and significantly positively correlated with age (r = 0.601, P < 0.01), years since menopause (r = 0.577, P < 0.01), systolic blood pressure (r = 0.295, P < 0.01), and diastolic blood pressure (r = 0.264, P < 0.05), but was not with other variables. In multivariate regression analysis, the baPWV was significantly correlated with BMD (P < 0.05), but not with other variables. CONCLUSIONS: Postmenopausal women with osteoporosis may have elevated arterial stiffness, suggesting that osteoporotic postmenopausal women may have a higher risk of cardiovascular disease.

Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells.

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).

Double strandedness of nascent DNA of a higher plant (Vicia faba).

After a pulse of 5-10 min with [3H]thymidine, labeled DNA extracted from embryonic axes of Vicia seeds sedimented as a rather homogeneous peak at approx. 10S in an alkaline sucrose density gradient, as described in our previous paper ((1975) Biochim. Biophys. Acta 395, 314-321). The sedimentation pattern of the same pulse-labeled DNA in a neutral gradient showed a wide range of sizes from approx. 14 S to more than 40 S. However, most of these labeled DNA components, including the 14-20 S shorter fragments, were shown to have a double-stranded structure by hydroxyapatite column chromatography. Further investigations on neutral sucrose gradients revealed a decreased occurrence after a chase, and final disappearance after a longer chase period of these shorter double-stranded fragments. A possible secondary structure of the newly synthesized DNA in a higher plant (Vicia faba) is discussed.

Rapidly labeled intermediates in DNA replication in higher plants.

In detached Vicia embryos, the incorporation of [3-H] thymidine into DNA starts at about 25 h after the beginning of imbibition and reaches maximum at about 33 h. The DNA synthesized during the first replication phase was extracted. Alkaline sucrose density-gradient analyses of the DNA indicated the occurrence of several short pieces of rapidly labeled DNA having sedimentation values of approx. 10 S and 14 S, after a pulse for 5 to 10 min. Prolonged labeling and chase incubation led to a shift of the shorter fragments to longer ones of 19 S and 22 S or more, thus indicating the nature of intermediates during DNA replication of these short fragments.

Association of HLA-DR, DQ genotype with different beta-cell functions at IDDM diagnosis in Japanese children.

Japanese IDDM patients have been demonstrated to have unique and different HLA associations from white patients. To elucidate the effect of HLA-associated genetic factors on the clinical heterogeneity of IDDM in Japanese people, HLA-DRB1, DQA1, and DQB1 genotypes in 88 childhood-onset Japanese IDDM patients were examined by polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) or sequence-specific primers (SSP). Of the 88 IDDM patients, 26 (29.5%) had DRB1*0405-DQA1*0302-DQB1*0401/X (DR4-DQ4/X), 38 (43.2%) had DRB1*0901-DQA1*0302-DQB1*0303/X (DR9-DQ9/X), and 9 (10.2%) were DR4/9-DQ4/9 heterozygous in the present study (X does not contain protective alleles). Clinical heterogeneity such as age distribution at onset, prevalence and serum level of anti-GAD antibodies (GADAb), and residual pancreatic beta-cell function after diagnosis were compared between patients with HLA-DR4-DQ4 and DR9-DQ9. The frequency of DR9-DQ9 genotype was significantly higher in the younger (0-10 years) than in the older (11-16 years) age-group of onset, but the frequency of DR4-DQ4 was higher in the older (11-16 years) age-group. Although no association of DR-DQ genotypes with the prevalence and serum level of GADAb was found among newly diagnosed patients, long-standing DR9-DQ9 patients had significantly higher levels of GADAb than those with DR4-DQ4. While no difference in time course of serum C-peptide (CPR) levels was detected between GADAb+ and GADAb- patients, a remarkable difference was demonstrated between DR9-DQ9 and DR4-DQ4 patients. The residual pancreatic beta-cell function was retained more in patients with DR4-DQ4 than in those with DR9-DQ9 at diagnosis through 12-18 months after diagnosis. These results suggest that the DR9-DQ9 genotype may induce stronger autoimmune destructive response (T-helper 1 function) against target beta-cells than the DR4-DQ4 genotype does. Our findings may warrant further studies on the association of diabetogenic autoimmune response with HLA class II molecules and contribute to a clarification of interracial differences in HLA-encoded susceptibility to IDDM.

Diagnosis of coronary stenosis by two-dimensional echographic study of dysfunction of ventricular segments during and immediately after pacing.

The adequacy of two-dimensional echocardiography during right atrial pacing for the detection and characterization of coronary artery stenosis was examined in 10 closed chest dogs. Pacing at successively higher rates up to 210 beats/min was carried out in the control state and again during a 70% left anterior descending coronary artery stenosis-induced with intracoronary plugs. Left ventricular short-axis echographic cross sections were obtained at several levels of the left ventricle. After computer-aided standardized subdivision, contractile function of the global section and its subsegments was characterized by computed systolic fractional area change percent and wall thickening percent. Ventricular segments supplied from the site of the 70% coronary stenosis were delineated in a low papillary level cross section by a myocardial contrast echographic technique, and these segments demonstrated significant dysfunction during pacing at 150 to 210 beats/min. Echographic observation of the involved segments immediately after pacing revealed a maximal depression of function 5 seconds after pacing, equivalent to dysfunction at peak pacing, with function returning to control levels within about 2 minutes. Both maximal pacing and early postpacing studies facilitated satisfactory discrimination of ischemic from normally perfused myocardial segments. These experiments show that right atrial pacing study with quantitative two-dimensional echocardiography may serve to detect and assess a coronary stenosis associated with minor or no cardiac dysfunction in the rest state.

Relation between myocardial injury and postextrasystolic potentiation of regional function measured by two-dimensional echocardiography.

An experimental study was designed to validate postextrasystolic potentiation assessment of myocardial viability or functional reserve of cardiac segments after acute coronary occlusion. Segmental systolic fractional area changes and wall thickening in pacing-induced postextrasystolic beats were mapped in 12 closed chest dogs by two-dimensional echocardiography during a control period and from 20 minutes to 3 hours after occlusion of the left anterior descending coronary artery. The extent of myocardial ischemic and necrotic zones was evaluated in left ventricular slices and subsegements corresponding to echographic cross sections. During two-dimensional echocardiography, left ventricular segments that were found to be neither ischemic nor necrotic always exhibited a significant augmentation of both fractional area change and wall thickening during the postextrasystolic beat that followed an induced premature contraction with a 42.4% coupling interval. In segments without necrosis but with varying degrees of ischemia, significant postextrasystolic potentiation was also demonstrated, even after 3 hours of occlusion. In contrast, segments that developed more than 80% necrosis failed to potentiate systolic fractional area change after 2 hours, and systolic wall thickening, even after 20 minutes of coronary occlusion. Statistical evaluation revealed a characteristic threshold at 41 to 60% necrosis, beyond which no potentiation of function could be elicited 3 hours after occlusion. Extrapolation from the experimental data suggests that when two-dimensional echographic studies in myocardial ischemia indicate postextrasystolic augmentation of segmental left ventricular function, the latter segments may be assumed to contain only small infarcts or to consist of reversibly ischemic and normal myocardium. Conversely, segments that fail to exhibit postextrasystolic potentiation can be assumed to be more than 60% necrotic.

Characterization of left ventricular mechanical function during arrhythmias by two-dimensional echocardiography. II. Location of the site of onset of premature ventricular systoles.

Two-dimensional echocardiography was applied experimentally in a closed chest dog model with intact pericardium to determine the location, magnitude and extent of contractile response during pacing from discrete ventricular sites. Midventricular short-axis tomographic images obtained during regular sinus rhythm and subsequent premature ventricular beats provided comparative measurements of global and segmental systolic changes of cross-sectional luminal areas and myocardial wall thickness. Computer-assisted standardized analysis of segmental systolic fractional area change and wall thickening was used to map left ventricular contraction during normal rhythm and premature beats of 70% coupling interval, induced alternately from anterior and lateral aspects of the mid-left ventricular short-axis cross-sectional plane. A characteristic pattern consisting of early systolic contraction and wall thickening was followed by paradoxical motion and wall thinning in late systole in segments corresponding to the region of direct electrical stimulation. Statistical analysis of segment by segment function indicated a maximal amount of premature beat contractile derangement at the site of the stimuli. Pacing from a right ventricular wall site in the midventricular plane caused a similar premature beat response at the anterior aspect of the interventricular septum. It is concluded that two-dimensional echographic analysis of segmental ventricular function can identify the location of electrical stimuli, and thus might noninvasively characterize regional patterns of contraction associated with ectopic foci during arrhythmias.

Verification of myocardial contrast two-dimensional echocardiographic assessment of perfusion defects in ischemic myocardium.

Myocardial contrast two-dimensional echocardiography was used in 21 closed chest dogs to assess its ability to delineate the extent of underperfused acutely ischemic myocardium. An agitated saline-Renografin echocardiographic contrast agent was injected into the left main coronary artery after left anterior descending coronary artery occlusion, and the size of the contrast echo-free area characterizing the perfusion defect was outlined in short-axis cross sections of the left ventricle. In 13 dogs, monastral blue dye was injected after 45 minutes of coronary artery occlusion and before sacrifice to provide anatomic delineation of underperfused zones in equivalent sections. Perfusion defects assessed by contrast two-dimensional echocardiography correlated well with those delineated by monastral blue dye (r = 0.91). Contrast echocardiographic study was also performed in eight other dogs at 5 hours of occlusion, after which infarct size was measured with triphenyl-tetrazolium-chloride. Contrast echocardiographic outline of the perfusion deficiency correlated but slightly overestimated the extent of necrosis (r = 0.88). It is concluded that contrast two-dimensional echocardiography can detect and outline the underperfused "risk area" during acute coronary artery occlusion, and may also permit assessment of the extent of myocardial infarction.

Correlation of myocardial echo contrast disappearance rate ("washout") and severity of experimental coronary stenosis.

The relation between experimental coronary stenosis and myocardial contrast echo disappearance rate ("washout") was investigated in anesthetized closed chest dogs. Of 13 dogs, 8 had serial contrast echographic studies with two successive degrees of coronary stenosis (50 and 70%) produced by threading stenotic plugs into the proximal left circumflex coronary artery. Studies were repeated with complete coronary occlusion achieved by inflation of an intracoronary balloon immediately proximal to the plugs. Myocardial contrast echograms were recorded in short-axis cross sections of the left ventricle after intracoronary injection of 2 ml hand-agitated saline-Renografin solution through a catheter placed in the coronary artery. An echo contrast washout index (t 1/2) was measured by digital processing computer analysis of successive end-diastolic images obtained by two-dimensional echocardiography during myocardial contrast agent injection. The injection to injection correlation coefficient of these t 1/2 measurements was satisfactory (r = 0.87, standard error of estimate 4.8 seconds). Involved segment t 1/2 measurements were found to be significantly altered by intracoronary stenosis and occlusion, ranging from 23 +/- 6 seconds (mean +/- standard deviation) in the control state, 29 +/- 9 and 44 +/- 10 seconds for 50 and 70% stenosis, respectively, and 104 +/- 35 seconds for total occlusion. It was concluded that myocardial contrast two-dimensional echocardiographic measurement of t 1/2 appears to be a useful index of the degree of coronary stenosis.

Enhancement in accessibility to macrophages by modification of mucin-type carbohydrate chains on a tumor cell line: role of a C-type lectin of macrophages.

We investigated the role of mucin-type (O-linked) carbohydrate chains of tumor target cells in the recognition by macrophages through a Gal/GalNAc-specific calcium-dependent lectin. Binding of a soluble form of this lectin to P815 mastocytoma cells was increased by treatment with benzyl-GalNAc, which presumably inhibited the extension of mucin-type carbohydrate chains. The levels of cell surface expression of GalNAc residues were elevated after benzyl-GalNAc treatment, as revealed by the binding of Vicia villosa agglutinin B4 and Dolichos biflorus agglutinin. Adhesion of treated P815 cells to this lectin immobilized on plastic surfaces also increased. Furthermore, the binding of P815 cells to macrophage-like RAW 264.7 cells and to peritoneal macrophages also increased after the same treatment. We concluded that elevated levels of cell surface terminal GalNAc in mucin-type carbohydrate chains increased accessibility of P815 cells to macrophages through Gal/GalNAc-specific calcium-dependent lectins.

Attenuated total reflection-Fourier transform infrared spectroscopy as a possible method to investigate biophysical parameters of stratum corneum in vivo.

We investigated the use of attenuated total reflection-Fourier transform infrared spectroscopy as a method to study differences in the molecular components of human stratum corneum in vivo. These variations as a function of the anatomic site and of the depth into its layered structure are important to understand the biology and physiology of the tissue. In this preliminary study we have investigated spectroscopic changes in 18 healthy individuals. Total reflection-Fourier transform infrared spectroscopy represents a potentially powerful tool to study biophysical properties of surfaces. We observed that, in vivo, biophysical parameters of the stratum corneum (such as hydration, lipid composition, and conformation of the aliphatic chains) are indeed dependent on the anatomic site. As in total reflection-Fourier transform infrared spectroscopy experiments the penetration depth of the evanescent field into the stratum corneum is comparable with the thickness of a layer of corneocytes, this technique can be used to follow the distribution of lipids, water, and proteins as a function of depth into the tissue. We found that, in vivo, these molecular components are non-uniformly distributed, in agreement with the presence of water and lipid reservoirs as observed with ex vivo ultrastructural investigations. Composition and conformational order of lipids are also a function of depth into the stratum corneum. Finally we compared the in vivo superficial hydration measured using the infrared absorption of the OH stretch of water, with the hydration measured using the Skicon hygrometer. Our results indicate that total reflection-Fourier transform infrared spectroscopy might be useful to measure important chemical and biophysical parameters of stratum corneum in vivo.

Effect of renal perfusion pressure on renal interstitial hydrostatic pressure and sodium excretion. Role of vasopressin V1 and V2 receptors.

Renal interstitial hydrostatic pressure (RIHP) has recently been cited as an important mediator of pressure natriuresis. Our objective was to determine the roles of vasopressin V1 and V2 receptors in mediating the effects of renal perfusion pressure (RPP) on RIHP and sodium excretion (UNaV). The effects of RPP on renal hemodynamics, RIHP, and UNaV were assessed in control Wistar rats (n = 10) and in rats pretreated with intravenous infusion of the specific nonpeptide vasopressin V1 antagonist OPC-21268 (100 micrograms.kg-1.min-1; n = 8) and the V2 antagonist OPC-31260 (40 micrograms.kg-1.min-1; n = 10). Increasing RPP from 95 to 118 mm Hg in control rats increased RIHP (6.4 +/- 1.0 to 9.9 +/- 1.3 mm Hg), UNaV (0.29 +/- 0.03 to 0.52 +/- 0.05 muEq.min-1.g-1), urine flow rate (UFR) (5.2 +/- 0.3 to 7.6 +/- 0.6 microL.min-1.g-1), and the fractional excretion of sodium (FENa). In rats pretreated with V1 antagonist, similar results were obtained for urine osmolality and the responses of RIHP, UNaV, UFR, and FENa to RPP. V2 antagonist reduced urine osmolality (392 +/- 47 compared with 979 +/- 88 mOsm.kg-1 in control rats) and enhanced the responses of UNaV (0.43 +/- 0.08 to 1.32 +/- 0.32 microEq.min-1), UFR (17.8 +/- 3.2 to 29.2 +/- 3.8 microL.min-1.g-1), and FENa to RPP, but the RIHP response resembled that observed in the control and V1 antagonist groups. Renal blood flow and glomerular filtration rate did not differ among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Humoral factor in pressor hyperresponsiveness in renal prehypertensive rabbits.

Prehypertensive rabbits with renal artery stenosis of 3 days' duration (one-kidney, one clip) are known to have increased pressor responses to norepinephrine and vasopressin; this pressor hyperresponsiveness is restored to normal by the angiotensin II (AII) antagonist, [ Sar1, Ile8 ] AII, even though plasma renin activity (PRA) and plasma AII concentrations are not elevated. In the present study, the cross-circulation of blood between conscious one-kidney, 3-day renal artery stenosis rabbits and conscious normal rabbits resulted in the transfer of pressor hyperresponsiveness to the normal rabbits, although both groups of rabbits had normal values for PRA. A similar cross-circulation of blood between one-kidney rabbits without renal artery stenosis and normal rabbits did not alter the pressor responsiveness of the normal rabbits to norepinephrine. It was concluded that a circulating humoral factor is involved in mediating pressor hyperresponsiveness in 3-day renal artery stenosis rabbits. To evaluate the interrelationship between AII and the hormonal hyperresponsiveness factor, an additional experiment was performed in which blood was cross-circulated between one-kidney, 3-day renal artery stenosis rabbits and normal rabbits, with the normal rabbits receiving [ Sar1, Ile8 ] AII immediately following cross-circulation. Administration of this AII antagonist to the normal rabbits prevented them from showing pressor hyperresponsiveness following the cross-circulation of blood. It is concluded that in this prehypertensive renal artery stenosis model the humoral hyperresponsiveness factor exerts its effect through AII mechanisms, rather than AII acting to increase the release or secretion of the hyperresponsiveness factor.

Pressor hyperresponsiveness in saline-infused rabbits.

Conscious rabbits infused intravenously (i.v.) with isotonic saline at 1.5 to 1.8 ml/min for 24 hours had greater pressor responses to norepinephrine (NE) than did normal control rabbits. Infusion of the angiotension II (ANG II) antagonist [Sar1, Ile8] ANG II did not decrease the exaggerated pressor responses to NE in saline-infused rabbits. Measurements of cardiac output (CO) as well as the pressor responses to NE before and after saline infusion revealed that, although saline infusion increased the CO and decreased total peripheral resistance (TPR), CO did not change during NE infusion either before or after saline infusion, but NE produced significantly greater increases in mean arterial pressure (MAP) and TPR after saline infusion than before the saline infusion. The cross-circulation of blood at 10 ml/min for 5 minutes between saline-infused donor rabbits and normal recipient rabbits resulted in pressor hyperresponsiveness to NE in the normal recipients. Similar cross-circulation experiments between pairs of normal rabbits did not alter the pressor responses to NE. These studies provided direct evidence that expansion of body fluid volumes by saline infusion results in pressor and vascular hyperresponsiveness. There was no evidence to indicate that ANG II was involved in the mechanisms producing this pressor hyperresponsiveness. Some circulating hormonal factor, however, was involved in mediating the pressor hyperresponsiveness following saline infusion. The results of this study are compatible with the concept that natriuretic hormone may play a role in promoting pressor hyperresponsiveness in saline-expanded animals.

Co-elevation of brain natriuretic peptide and proprotein-processing endoprotease furin after myocardial infarction in rats.

We investigated the expression of the yeast Kex2 family endoproteases furin and PACE4, and brain natriuretic peptide (BNP) in the atrium and ventricle after infarction as well as the conversion of the BNP precursor gammaBNP to BNP-45. In a rat heart failure model, plasma BNP rose in two phases--first at day 3, and again at day 14. BNP mRNA, as measured by Northern blot analysis, increased strongly at day 3, then at days 14 and 28 less strongly in the atrium, and in the ventricle it increased weakly at day 3, then strongly at days 14 and 28. Furin mRNA showed the same pattern of expression as that of BNP message, whereas PACE4 message stayed unchanged after the infarction. Both furin and BNP were immunostained in the myocardium adjacent to the infarcted tissue. We suggest that after myocardial infarction, furin is co-expressed with BNP in both the atrium and ventricle, and that furin may be responsible for the conversion of gammaBNP to BNP-45.

Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

Serum level of vascular endothelial growth factor is decreased by hormone replacement therapy in postmenopausal women without hypercholesterolemia.

The administration of hormone replacement therapy (HRT) to postmenopausal women (PMW) reportedly has beneficial effects on their levels of lipid and lipoproteins. Estrogen retards the development of atherosclerosis induced by a high-fat diet in animals. Although vascular endothelial growth factor (VEGF) may be involved in the development of atherosclerosis in humans, there is no information on effect of estrogen administration on VEGF level and lipid metabolism. We evaluated 64 healthy normotensive or hypertensive PMW before and during the administration of HRT (0.625 mg conjugated equine estrogen combined with 2.5 mg medroxyprogesterone acetate orally) daily for 6 months. All hypertensive PMW were well-controlled on antihypertensive drug therapy. According to their total cholesterol level at baseline, we divided the PMW with HRT (n=54) into a normocholesterolemic group (NC, total cholesterol <220 mg/dl, n=35) and a hypercholesterolemic group (HC, total cholesterol >/=220 mg/dl, n=19). We evaluated the serum levels of VEGF at baseline, and again at 3 and 6 months after starting HRT. HRT significantly (P<0.01) reduced the mean VEGF level from 31.5+/-4.3 pg/ml at baseline to 18.2+/-2.3 pg/ml after 6 months in the NC group. However, the VEGF levels in the HC group and the control group exhibited no significant change at either 3 or 6 months after starting HRT. In summary, HRT, using a combination of conjugated equine estrogen and medroxyprogesterone acetate, reduced the level of VEGF in normocholesterolemic PMW more effectively than in those with hypercholesterolemia.