Mint3-mediated L1CAM expression in fibroblasts promotes cancer cell proliferation via integrin α5ß1 and tumour growth.

Fibroblasts are some of the major cells in tumour tissues that influence tumour progression and drug resistance. However, our understanding on fibroblast-mediated tumour malignancy remains incomplete. Munc18-1-interacting protein 3 (Mint3) is known as an activator of hypoxia-inducible factor-1 (HIF-1) even during normoxia in cancer cells, macrophages and fibroblasts. Although Mint3 promotes ATP production via glycolysis by activating HIF-1 in cancer cells and macrophages, the biological role of Mint3-mediated HIF-1 activation in fibroblasts remains unclear. To address this, we examined whether Mint3 in fibroblasts contributes to tumour growth. Mint3 depletion in mouse embryonic fibroblasts (MEFs) decreased tumour growth of co-injected human breast cancer cells, MDA-MB-231 and epidermoid carcinoma A431 cells in mice. In MEFs, Mint3 also promoted cancer cell proliferation in vitro in a cell-cell contact-dependent manner. Mint3-mediated cancer cell proliferation depended on HIF-1, and further gene expression analysis revealed that the cell adhesion molecule, L1 cell adhesion molecule (L1CAM), was induced by Mint3 and HIF-1 in fibroblasts. Mint3-mediated L1CAM expression in fibroblasts stimulated the ERK signalling pathway via integrin α5ß1 in cancer cells, and promoted cancer cell proliferation in vitro and tumour growth. In cancer-associated fibroblasts (CAFs), knockdown of MT1-MMP, which promotes Mint3-mediated HIF-1 activation, or Mint3 decreased L1CAM expression. As MEFs, CAFs also promoted cancer cell proliferation in vitro, and tumour growth via Mint3 and L1CAM. In human breast cancer specimens, the number of fibroblasts expressing L1CAM, Mint3 and MT1-MMP was higher in cancer regions than in adjacent benign regions. In addition, more phospho-ERK1/2-positive cancer cells existed in the peripheral region surrounded by the stroma than in the central region of solid breast cancer nest. Thus, Mint3 in fibroblasts might be a good target for cancer therapy by regulating cancer cell-stromal cell communication.

Genome-wide association study of degenerative bony changes of the temporomandibular joint.

OBJECTIVES: To identify susceptibility genes underlying degenerative bony changes of the temporomandibular joint (TMJ). MATERIALS AND METHODS: Bony changes of the TMJ condylar head were diagnosed by examination of panoramic radiographs and/or magnetic resonance images and/or computed tomography images. We conducted a genome-wide association study (GWAS) of 146 cases with TMJ degeneration and 374 controls from East Asian populations using an Illumina HumanOmniExpress BeadChip. After rigorous quality-control filtering, approximately 550,000 single nucleotide polymorphisms (SNPs) were used for tests of associations with disease status. RESULTS: Forty-one SNPs at 22 independent loci showed association signals at P < 1 × 10(-4). The SNP rs878962, which maps on an intron of TSPAN9 on chromosome 12, showed the strongest association (combined OR = 1.89, 95% confidence interval = 1.43-2.50, P = 8.1 × 10(-6)). According to in silico predictions of the 41 SNPs, two intronic SNPs of APOL3 (rs80575) and MRC2 (rs2460300) may fall within regulatory elements and affect DNA-protein interactions. We could not replicate SNPs located on genes that have been reported to be associated with temporomandibular disorder or temporomandibular osteoarthritis in previous studies at P < 1 × 10(-4). CONCLUSIONS: Our GWAS identified 22 independent loci showing suggestive association signals with degenerative bony changes of the TMJ. These loci provide good candidates for future follow-up studies.

Positional effects of polymorphisms in probe-target sequences on genoplot images of oligonucleotide microarrays.

Single nucleotide polymorphisms (SNPs) present in probe-target sequences (SPTS) have been shown to be associated with abnormal genoplot images. We explored the effects of SPTS positions on genoplot images using a data set from a genome-wide association study typed on an Illumina Human Hap300 platform. We screened the physical genomic positions of 308,330 autosomal probes to identify SPTS candidates deposited in dbSNP. The genoplot images across 293 individuals were inspected further in SNPs bearing an SPTS candidate. We identified 35,185 SNPs bearing a single SPTS candidate, including 264 SNPs showing abnormal genoplot images. The frequencies of SPTS at distances within 10 bases from the target SNP were significantly higher in the 264 SNPs showing abnormal genoplot images, than in the remaining 34,921 SNPs (49.62 vs 12.87%; Fisher exact test; P = 2.2 x 10(-16)). Of these 264 SNPs, we randomly selected 20 SNPs and resequenced them in 97 individuals. An SPTS within 10 bases of the target SNP was confirmed in all 20 SNPs, except for one SNP with a small deletion (7 bases) in the probe-target sequence. Taken together, these results suggest an association of a proximal SPTS with an abnormal genoplot image, which could result in spurious genotype detections, highlighting the importance of minimizing systematic errors in microarray experiments.

The use of link provider data to improve national genetic evaluation across weakly connected subpopulations.

Data from 3 prefectures and a nationwide farming corporation were used to assess the usefulness of the "link provider data" in providing indirect genetic links for the national genetic evaluation for carcass weight across weakly connected subpopulations of the Japanese Black cattle. The data from the farming corporation provided genetic links to those of all prefectures and was therefore used as the link provider data. Two national genetic evaluation strategies under an animal model were compared, based on the generalized coefficient of determination (CD) of contrasts between mean EBV of sires or maternal grandsires (MGS) from different prefectures: strategy PA-1 was a pooled analysis of the data sets of the 3 prefectures, and strategy PA-2 was a pooled analysis of the data sets of the 3 prefectures and the farming corporation. The CD of the contrasts were greater for PA-2 than for PA-1. Under PA-2, the CD of the contrasts between mean EBV of sires or MGS ranged from 0.67 to 0.78 or from 0.61 to 0.70, respectively. Pooling the data from the 3 prefectures and the farming corporation increased the degree of connectedness through the link provider data rather than the amount of information by adding more data, thus improving the accuracy of prediction. The differences between mean EBV of sires or MGS from different prefectures were smaller for PA-1 than for PA-2. This finding suggests that genetic differences in carcass weight among prefectures are present, but that they would be confused with the environmental differences under PA-1 because of the lack of genetic connectedness among the prefectures. On the other hand, the genetic differences among the prefectures would be predicted precisely under PA-2 because the genetic connectedness among the prefectures was improved by using the link provider data. The results demonstrate that the link provider data could be used to unify within-prefecture evaluation to form a Japanese national genetic evaluation across weakly connected subpopulations.

Estimation of heritability and genetic correlation for behavioural responses by Gibbs sampling in the Thoroughbred racehorse.

Genetic variation of the behaviour of racehorses is one of the major concerns for racehorse breeders. In this study, the heritabilities of behavioural responses to the inspections of conjunctiva, auscultation and blood sampling and the genetic correlations among them were estimated in the Thoroughbred racehorse. The estimation was done with Bayesian analysis with Gibbs sampling based on the univariate or bivariate threshold animal models. The behavioural responses were scored with four categories at the first entrance quarantine in Miho Training Center of Japan Racing Association from 1993 to 1995. The behavioural responses were treated as categorical or binary traits, with both showing similar results. The estimated heritabilities were in the range of 0.23-0.28, suggesting a genetic component in the variation on these traits. The estimated genetic correlations among the traits were very high (approximately 0.9), suggesting that these behavioural responses may be measures of the same trait. Because of the high genetic correlations, repeatability threshold model was applied assuming the responses to be a genetically identical trait measured with three different tests. The estimated heritabilities (approximately 0.23) were at the lower bound of the former estimates. The revealed high repeatabilities (0.97-0.98) suggest a strong contribution of the individual temperament on the behaviour of racehorses.

Effectiveness of adjusting for heterogeneity of variance in genetic evaluation of Japanese Black cattle.

Heterogeneity of variance among subclasses of an effect is a potential source of bias in genetic evaluation. The objectives of this study were to quantify the heterogeneity of variance in carcass weight in Japanese Black cattle, to develop an adjustment method to account for the heterogeneity, and to evaluate the effectiveness of the method. A total of 96,950 records were collected from steers and heifers slaughtered from 1997 to 2005. These records were grouped into 2,767 farm-market-year-sex subclasses. Fourteen log-linear models for the variances were set up to estimate the heterogeneous phenotypic variances within subclasses. Schwarz's Bayesian information criterion was used for model selection. The preadjustment of records to a baseline variance was based on maximum likelihood estimates obtained from the selected model. As a result of adjustment, the SD, the CV, and the Gini coefficient for the phenotypic variance decreased by 68.6, 69.8, and 70.1%, respectively. When the top 5% of sires and top 1% of dams were selected, Spearman's rank correlation coefficients between the adjusted and unadjusted data were 0.95 for the selected sires and 0.78 for the selected dams. The effectiveness of the adjustment was evaluated in terms of the ability to predict breeding values, using the results of the successive genetic evaluations. Mean squared error between the parent averages and actual predicted values of the genetic merit for the sires whose progeny had a carcass record only from 2003 to 2005 was significantly reduced by the adjustment (P < 0.05). The results suggest that the genetic evaluation becomes more accurate by adjusting the data using the procedure developed in this study.

Development of a real-time hand dose monitor for personnel in interventional radiology.

Medical procedures denoted as interventional radiology require operation near an X ray beam, which brings high dose exposures to the operators' hands. For the effectual control of their extremity doses, a prototype of a real-time wrist dosemeter has been developed, hand dose monitor (HDM), based on a single silicon detector. Experiments were performed to test its response to diagnostic X rays. The HDM was highly sensitive and showed a linear response down to doses of a few tens of microsieverts. Though dose rate, energy and angular dependence of the response were observed in some extreme conditions, the HDM was proved to be of practical use if it was appropriately calibrated. Since an HDM enables personnel to check their hand doses on a real-time basis, it would enable medical staff to control the exposure themselves.

[Assessment of hydroxylated metabolites of polychlorinated biphenyls and polychlorinated dibenzofurans as potential estrogens by yeast two-hybrid system].

The estrogenic activities of several hydroxylated metabolites of PCBs and PCDFs were investigated by yeast two-hybrid assay based on the ligand-dependent interaction of estrogen receptor with coactivator. For the hydroxylated PCBs, the order of estrogenic potency was 4-OH-2',4',6'-triCB > 4-OH-4'-monoCB, 4-OH-biphenyl. These compounds were evaluated as 10(3) to 10(4) less potent than 17 beta-estradiol based on the concentrations of test compounds showing 10% activity of 10(-7) M 17 beta-estradiol. 2-OH-3',4,4'-triCB, 4-OH-2',3,4'-triCB and 3-OH-/4-OH-2,2',5,5'-tetraCB, the metabolites of 2,2',5,5'-tetraCB were inactive as estrogens at the highest concentrations used in this study (10(-5) M). Also 4-OH-3,3',4',5-tetraCB, the metabolite of 3,3',4,4'-tetraCB was inactive as estrogen, indicating that this hydroxylated metabolite did not take part in the estrogenic activity of 3,3',4,4'-tetraCB. OH group at 4-position of biphenyl was necessary for the expression of estrogenicity, but one or two chloro-substitution adjacent to OH group inhibited the activity. For the hydroxylated PCDFs, 8-OH-2-monoCDF, 7-OH-3,4-diCDF, 8-OH-3,4-diCDF, 8-OH-3,4,6-triCDF and 3,8-(OH)2-2-monoCDF exhibited estrogenic activity. The estrogenic activity of 3,8-(OH)2-2-monoCDF was comparable to those of 4-OH-2',4',6'-triCB and 4-nonylphenol (mixture of compounds with branched sidechain). The order of activity was 3,8-(OH)2-monoCDF > 8-OH-3,4-diCDF, 7-OH-3,4-diCDF > 8-OH-2-monoCDF, 8-OH-3,4,6-triCDF. These compounds were evaluated as 2.5 x 10(3) to 3 x 10(4) less potent than 17 beta-estradiol. On the other hand, no estrogenic activity was observed for 2-OH-dibenzofuran, 3-OH-2,8-diCDF, 6-OH-3,4-diCDF and 9-OH-3,4-diCDF at concentrations as high as 10(-4) M. Substitution of OH group at 2(8)- or 3(7)-position of dibenzofuran and no chloro-substitution adjacent to OH group was required for the estrogenic activity.

[Behavior of Trp-P-1 and its metabolites in rat excreta].

To study of the behavior of Trp-P-1 and its metabolites in rat feces and urine, rats were orally administered with Trp-P-1 (750, 1,500 and 2,500 micrograms/rat), and excreted Trp-P-1 was analyzed using HPLC assay and bacterial mutagenicity assay. The extraction of Trp-P-1 from urine was performed by using the chloroform extraction method, and blue rayon was used for the extraction from feces. When Trp-P-1 was added to rat feces and urine, the recoveries of Trp-P-1 were 85.9 +/- 3.9% and 91.3 +/- 3.7%, respectively. The extracts of feces and urine from rats administered with Trp-P-1 were individually fractionated by thin layer chromatography on C18 gel. The major mutagenic zone corresponding to Trp-P-1 was found at Rf 0.09 in both extracts, while the feces extract gave two additional mutagenic zones at Rf 0.15 and 0.20. More than 97% of the fecal mutagenic activity was due to unchanged Trp-P-1. In rats administered with 750 micrograms of Trp-P-1, the amount of extracted Trp-P-1 and the number of His+ colonies induced by whole excreta were 81.6 +/- 7.1 micrograms (n = 6) and (432 +/- 77) x 10(4) for feces, and 28.7 +/- 4.9 micrograms and (171 +/- 28) x 10(4) for urine. The recoveries of Trp-P-1 in the feces and urine were 10.8 +/- 0.9% and 3.8 +/- 0.7% by HPLC analysis, and 11.1 +/- 2.0% and 4.4 +/- 0.7% by mutagenicity assay respectively. The results of the two assays seemed to show similar patterns of recovery.

Entire scalp replantation: case report and review of the literature.

A case of successful replantation of a totally avulsed scalp in a 40-year-old woman is presented. During a 19-hr surgical procedure, bilateral superficial temporal arteries and veins were anastomosed. The postoperative course was uneventful, except for partial necrosis of the distal end of the avulsed flap, i.e., the occipital region. Since the first successful scalp replantation using microsurgical technique was reported in 1976, the authors have found at least 32 subsequently reported cases. Of these, there were 22 cases of entire scalp avulsion (more than 80 percent of the scalp), including 15 females and seven males. A summary of these cases is included. From analysis of the reported cases of entire scalp avulsion, the keys to success in scalp replantation are considered to be the adequate selection of vessels for repair and the use of vein grafts, if necessary.

Proliferating activity of dermal fibroblasts in keloids and hypertrophic scars.

Monoclonal antibodies were used to demonstrate proliferating cell nuclear antigen (PCNA) and Ki-67 antigen of dermal fibroblasts in formalin-fixed, paraffin-embedded tissue sections of keloids, hypertrophic scars and normal skin. PCNA-stained fibroblasts were more pronounced than Ki-67, which showed only scanty Ki-67-positive fibroblasts. The mean density of dermal fibroblasts was significantly higher in keloids and hypertrophic scars than in normal skin (p < 0.01). The proliferating activity of fibroblasts detected by PCNA was significantly higher in keloids than in hypertrophic scars or normal skin (p < 0.01). The mean densities and the proliferating activities were apparently not correlated with the age of the patient. This indicates that keloids, with higher density and proliferating activity of dermal fibroblasts, continue to increase their volume and invade the surrounding tissue, while hypertrophic scars, with higher density and lower proliferating activity, show a tendency towards spontaneous regression.

Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

Boronate affinity chromatography of gamma-glutamyltransferase in patients with hepatocellular carcinoma.

We analyzed the serum gamma-glutamyltransferase (gamma-GT) by boronate affinity chromatography to ascertain the presence or absence of any changes in the binding properties of gamma-GT toward boronate gels in patients with hepatocellular carcinoma and liver cirrhosis, and in normal controls. The mean gamma-GT activity ratio of the bound (peak 2) and nonbound (peak 1) fraction in patients with hepatocellular carcinoma was significantly higher than that in patients with liver cirrhosis or in normal controls. Thus, the gamma-GT, which has adjacent cis-hydroxyl groups in its carbohydrate moieties, was found to increase in the serum of patients with hepatocellular carcinoma. The positivity rate was examined in patients with hepatocellular carcinoma and liver cirrhosis, using a cut-off level for the peak 2:peak 1 ratio of 1.05 (mean + 2 SD of liver cirrhosis). Nineteen (42.2%) patients with hepatocellular carcinoma had a ratio of peak 2:peak 1 higher than 1.05. Nine of the 19 patients who had serum alpha-fetoprotein levels below 100 ng/ml had an elevated peak 2:peak 1 ratio. In total, 77.8% of the occurrence of hepatocellular carcinoma could be detected by a combination of these two markers. Three patients who had developed hepatocellular carcinoma during the course of cirrhosis but remained negative for alpha-fetoprotein throughout the course developed higher levels of peak 2:peak 1 ratio when hepatocellular carcinoma occurred. These results indicate that the two markers, the peak 2:peak 1 ratio of serum gamma-GT activity and serum alpha-fetoprotein level, may be considered to serve as complementary markers for the diagnosis of hepatocellular carcinoma.

Discrimination of liver cirrhosis from chronic hepatitis by analysis of serum cholinesterase isozymes using affinity electrophoresis with concanavalin A or wheat germ agglutinin.

Isozymic alteration of serum cholinesterase (ChE) was investigated in patients with chronic liver diseases using affinity electrophoresis with concanavalin A (Con A) or wheat germ agglutinin (WGA). On Con A-containing agarose gel electrophoresis, three bands with enzyme activity (named bands I to III, from the anodic side to the cathodic) were observed in sera of normal controls. Disappearance of band II was observed in 50% (15/30) of cirrhotic patients, but only one of 20 patients with chronic hepatitis lacked band II of the serum ChE isozymes. Meanwhile, WGA-containing agarose gel electrophoresis revealed that normal controls had four ChE isozymes (named bands I to IV from the anodic side to the cathodic). These four isozymes were also observed in patients with chronic hepatitis. However approximately 67% (20/30) of cirrhotic patients lacked band II of ChE isozymes. When these two affinity electrophoreses were used in combination, 22 (73%) of 30 cirrhotic patients had isozymic alteration of their serum ChE on either Con A-containing or WGA-containing agarose gel electrophoresis, or both. Thus, affinity electrophoreses with Con A and WGA seemed to be useful methods in differentiating liver cirrhosis from chronic hepatitis.

Bilateral vermilion flaps for lower lip repair.

A more natural reconstructive procedure of the lower lip using bilateral vermilion flaps was applied in five patients with excellent results. The vermilion defects were about two-fifths to three-fifths. In three patients, the vermilion defect was repaired using bilateral vermilion flaps alone. In the remaining two patients, a narrow horizontal lip defect was repaired by bilateral vermilion flaps and a subcutaneous V-Y advancement flap of the lower lip. A single vermilion flap or bilateral vermilion flaps are considered to be of great value for vermilion reconstruction because of the inherent elasticity and common anatomic unit. The postoperative scars are not remarkable at all. A long and narrow horizontal lip defect (perhaps within 1.5 cm downward from the vermilion border) may be effectively repaired by the combination of vermilion flap(s) and a V-Y advancement flap without sacrificing any additional healthy tissue.

Age-related reduction in the number of rabbit erythrocyte Na, K-ATPase.

The number of Na, K-ATPase units, assessed by [3H]-ouabain binding assay, was significantly less in erythrocytes from 17 mature (30-week) rabbits than in those from 17 young (16-week) ones (0.179 +/- 0.010 vs. 0.263 +/- 0.014 pmol/10(9) cells, p less than 0.01). Consistent with this finding, the erythrocyte sodium concentration was significantly higher in mature rabbits as compared with young animals (17.1 +/- 1.30 vs. 10.8 +/- 0.68 mmol/liter, p less than 0.01). When the assays were repeated in 8 of the young rabbits 8 weeks after the initial determination, both variables were found to be similar to the levels from mature animals. The data suggest that the activity of erythrocyte sodium pump declines with aging or growth of donor rabbits, as a result of a reduction in the number of Na, K-ATPase units.

Quantification of myocardial infarct size after coronary reperfusion by serum cardiac myosin light chain II in conscious dogs.

The effects of early coronary artery reperfusion on the relation between the extent of myocardial infarction and serum levels of cardiac myosin light chain II or plasma creatine kinase levels were evaluated in the conscious dog. Hydraulic occluders were placed on the left anterior descending arteries of 38 dogs. Seven to 10 days later, myocardial infarction was produced. Coronary reperfusion was performed 3 hours (group A1, n = 13) and 6 hours (group A2, n = 12) after the occlusion. In the other 13 dogs, coronary occlusion was sustained throughout the course of the experiment (group B). Seven days after the occlusion, the heart was cut from the apex to the base into 4-mm slices, and infarct size was determined macroscopically. Rapid appearance and early peaking of creatine kinase were observed in group A. Cumulative release of creatine kinase significantly correlated with infarct size in group A (infarct size ranged from 0.1 to 20.1 g, r = 0.90) and group B (from 0.6 to 26.8 g, r = 0.91). However, since creatine kinase release in group A was greater in comparison with that from infarcts of the same size in group B, the slope of the regression line for group A was significantly steeper (p less than 0.05). Cardiac myosin light chain II appeared as early as creatine kinase did and continued to be elevated for 7 days. A very close relation was observed between infarct size and total cardiac myosin light chain II release (r = 0.87 for group A, and r = 0.88 for group B) or peak level of light chain II (r = 0.85 for group A, and r = 0.81 for group B). In addition, the slopes of the regression lines for infarct size and both peak and total release of light chain II did not differ between group A and group B. On histological examination, viable myocardium was frequently observed in the epicardium of the ischemic area in group A1; therefore, infarct size was greater in group B than in group A1 (p less than 0.05). Also, myocardial creatine kinase content in the epicardium of the center of the ischemic area in group A1 was greater than that in group B. Cardiac myosin light chain II release in group A1 was less than that in group B, whereas no difference was found in plasma creatine kinase release among groups A1, A2, and B.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum pseudouridine as a biochemical marker in patients with hepatocellular carcinoma.

The concentration of serum pseudouridine, a degradation product of transfer ribonucleic acid, was determined by high-performance liquid chromatography in patients with hepatocellular carcinoma, liver cirrhosis, other benign hepatobiliary diseases, and healthy controls. The serum pseudouridine concentration in patients with hepatocellular carcinoma was significantly higher than that in patients with cirrhosis or the controls. Twenty-seven (51.9%) of 52 patients with hepatocellular carcinoma had serum pseudouridine concentrations higher than the mean value for healthy controls plus 2 SD. Fourteen of the 36 patients who had serum alpha-fetoprotein levels below 400 ng/ml, had elevated serum pseudouridine concentration. In total, 36 of the 52 patients (69.2%) could be detected by combination of these two markers. Two patients who had developed hepatocellular carcinoma during the course of cirrhosis and were continuously negative for alpha-fetoprotein, had higher levels of the pseudouridine concentration when hepatocellular carcinoma occurred. Furthermore, 4 of the 7 patients who had a very small cancer and were negative for alpha-fetoprotein, had elevated serum pseudouridine concentration. These results indicate that serum pseudouridine is a useful biochemical marker and that serum pseudouridine and alpha-fetoprotein in combination are considered to serve as complementary markers, for the diagnosis of hepatocellular carcinoma.

Effects of monotherapy and withdrawal of antihypertensive drugs in the treatment of hypertension.

The final number of antihypertensive drugs used in the long-term treatment of a given patient is not always predictable at the start of therapy. By reviewing clinical records, we retrospectively examined the relationship between pretreatment blood pressure and the final number of drugs administered in 282 patients with mild to moderate hypertension who had been treated for 5 years or more (average 9.7 years). After years of treatment approximately one third and one half of 137 patients with a pretreatment diastolic blood pressure of between 90 and 104 mmHg were well controlled with combined therapy and monotherapy, respectively. The drugs had been withdrawn in the remaining 17% for 12 months or more. Lower pretreatment systolic blood pressure and lower pretreatment QRS voltage were signs favorable for withdrawal of the drugs. Combined therapy was required in more than half the patients with higher pretreatment diastolic blood pressure.