A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

Determination of percent body fat by the newly developed sulfur hexafluoride dilution method and air displacement plethysmography.

The reliability and validity of two newly developed densitometric methods for determining the human body volume and percent body fat (%FAT), the sulfur hexafluoride dilution method (SHF) and air displacement plethysmography (ADP), were evaluated in comparison with the underwater weighing method (UWW). Seven healthy male volunteers (age 31 to 44, mean height 166.0 cm, weight 61.4 kg) participated in this study. The same-day test-retest coefficients of variation (CVs) for body volume and %FAT measurements were not significantly different among the three methods. SHF and UWW showed a strong correlation in terms of body volume and %FAT, with the correlation coefficients (r) being 0.9997 and 0.986, respectively. The correlation between ADP and UWW was slightly weaker (r = 0.9997 for body volume and 0.907 for %FAT). However, body volumes measured by SHF and ADP were significantly different from that by UWW when compared by mean values. Such differences were also found for %FAT measurements. The regression lines of body volume measured by SHF and ADP on that by UWW were almost equivalent to the line of identity. However, those of %FAT measured by SHF and ADP on that by UWW were significantly different from the line of identity. Because the reliability of SHF and ADP appeared to be high, further validation and improvement are required and worth doing.

A novel isoform of rat estrogen receptor beta with 18 amino acid insertion in the ligand binding domain as a putative dominant negative regular of estrogen action.

A novel isoform of rat estrogen receptor (ER) beta, ER beta 2, which is a putative alternative splicing product of the reported ER beta (ER beta 1) has been identified. Rat ER beta 2 cDNA contains an additional, in-frame 54 base pair insertion in the ligand binding domain of ER beta 1, which generates an 18 amino acid residue insertion. Northern blot and RT-PCR analyses revealed that ER beta 2 coexists with ER alpha and ER beta 1 in all tissues examined including brain, lung, liver, kidney, fat, bone, uterus, prostate, and ovary. The insertion caused loss of ligand binding activity of ER beta 2, whereas the ability to bind the palindromic estrogen response element (ERE) was retained. In an ERE-containing luciferase reporter gene assay using COS-1 cells, ER beta 2 failed to activate estrogen-dependent transcription. Furthermore, ER beta 2 dose dependently suppressed the ER alpha- and ER beta 1-mediated transcriptional activation. These results suggest that rat ER beta 2 functions as a negative regulator of estrogen action.

Characteristics of energy metabolism in males with mental retardation.

To characterize the energy metabolism in individuals with mental retardation (MRs), we measured energy cost at several physical activity levels (basal, supine, sitting, standing, and walking at 30, 50 and 70 m/min), maximal oxygen consumption (Vo2max), and body composition in 23 male MRs and the same number of volunteer male controls. Both groups were individually matched for age, body height, and body weight. Energy cost was measured by the Douglas bag technique. The recently developed sulfur hexafluoride (SF6) dilution technique was employed for measuring body composition. In addition, 3-dimensional accelerometry was used for evaluating body movements, and plasma indices of macronutrients were also measured. The energy cost of MRs, when sitting, standing, and walking at 30 and 50 m/min, was significantly higher than that of controls (p < 0.05), while the basal and resting metabolic rates were similar in both groups. Vo2max was significantly lower (p < 0.05) in MRs than controls. Accelerometry demonstrated excessive movement by MRs, which may explain their higher energy cost of exercise. In contrast, no significant difference was observed in percent body fat or lean body mass. Concentrations of plasma total cholesterol, triacylglycerols and albumin were significantly lower in MRs as compared with the controls. Our findings suggest that MRs are burdened with an energy metabolism less economical than non-MRs. Limited physical activity in their daily life may be the cause. These characteristics of MRs' energy metabolism should be considered for planning their proper dietary schedules and physical activity programs.

A brief review of epidemiological studies on ischemic heart disease in Japan.

The age-adjusted death rate from ischemic heart disease in Japan is the lowest among developed countries and the rates have decreased since 1970. The incidences of myocardial infarction in selected populations ranged between 0.12 and 2.56 per 1,000 for middle-aged males, and between 0.00 and 1.52 per 1,000 for females. The incidences of sudden death within 24 hours were from 0.00 to 1.58 per 1,000 for males and from 0.00 to 0.76 per/1,000 for females. The incidences in Japanese populations appeared to be far below those in Western populations. In the Cox proportional hazard regression model, hypertension and smoking were selected as independent risk factors for myocardial infarction in an agricultural district. It was noteworthy that the level of serum cholesterol was not associated with development of myocardial infarction in rural areas. No positive relation between dietary fat and serum cholesterol was observed in school children, suggesting that growth, sexual maturation and others might be confounding variables between them. The levels of serum cholesterol for females were more affected by menopause than those of blood pressures and body mass index. Although some polymorphisms in selected candidate genes appeared to be associated with some serum lipids and apolipoproteins, the effect of individual RFLP on the inter-individual variations in serum traits was relatively subtle in comparison with that of lifestyle factors.

Detection of an amino acid polymorphism in hormone-sensitive lipase in Japanese subjects.

Hormone-sensitive lipase (HSL) plays an important role in energy metabolism by controlling the hydrolysis of triglycerides stored in adipose tissue. To investigate whether mutations in the HSL gene are associated with non-insulin-dependent diabetes mellitus (NIDDM), we screened for mutations of this gene using single-stranded conformation polymorphism (SSCP) in 35 Japanese subjects with NIDDM. SSCP analysis identified a variant pattern in axon 4, and the sequence showed that this variant pattern resulted from amino acid polymorphism (Arg309Cys). Subsequent study showed that this polymorphism was found in 18 of 151 NIDDM patients and 10 of 97 nondiabetic subjects, but allele frequency was not significantly different between the two groups (P = .7). Body mass index, serum triglyceride, and high-density lipoprotein (HDL) cholesterol were not different in subjects with and without the polymorphism. But serum total cholesterol was higher in subjects with the polymorphism than in subjects without it (P = .0005). These data indicate that this HSL polymorphism is not associated with NIDDM, obesity, and serum triglyceride level. However, an effect of the polymorphism to elevate serum total cholesterol has not been excluded, although further study is necessary to resolve its association with cholesterol metabolism.

Identification of two novel amino acid polymorphisms in beta-cell/liver (GLUT2) glucose transporter in Japanese subjects.

The beta-cell/liver glucose transporter (GLUT2) gene was screened for mutations using single-strand conformation polymorphism analysis (SSCP) in 30 Japanese subjects with non-insulin dependent diabetes mellitus (NIDDM). Analysis of all exons and adjacent intron regions identified six SSCP polymorphisms, three of which resulted in amino acid substitutions: V101I, T110I and G519E. The V101I and G519E, substitutions represent new polymorphisms in this gene. The six polymorphisms were observed in both NIDDM and control groups and there were no significant differences in allele frequencies between groups. A portion of the insulin receptor substrate 1 gene in 30 NIDDM subjects and in normal control subjects was also screened for mutations. Two SSCP variants that change the sequence of the protein, delta S686/687 (deletion of the codons for serine-686 and 687) and G972R, were identified in two different NIDDM subjects, both whom were also heterozygous for the V101I polymorphisms in GLUT2. The GLUT2 and IRS1 amino acid polymorphisms did not show a simple pattern of co-inheritance with NIDDM in the families of these subjects suggesting that neither polymorphism is sufficient to cause NIDDM but may increase diabetes-susceptibility through their interaction with other loci and environmental factors.

First-phase insulin response to glucose in nonobese or obese subjects with glucose intolerance: analysis by C-peptide secretion rate.

This study was proposed to clarify the impairment of first-phase insulin response to glucose in subjects with glucose intolerance by analysis of C-peptide secretion rate after glucose or glucagon injection. The rate was calculated from kinetic analysis of peripheral C-peptide behavior. The rate reached the peak two minutes after glucose injection and then rapidly declined (first-phase secretion) in control subjects. In nonobese subjects with impaired glucose tolerance (IGT) or non-insulin-dependent diabetes mellitus (NIDDM), the rate promptly increased in response to glucose and was followed by a second phase increase. The time course of the rate in the subjects was slightly different from that in control subjects. There was a progressively greater deficit in the first-phase increase with increasing severity of glucose intolerance. The time course of the rate in the obese subjects with NIDDM was different from that in control subjects. The first-phase increase was reduced in the obese subjects with NIDDM. The glucose disappearance rate was correlated with the first-phase increase. Since the time course of the rate after glucagon injection in all subjects did correspond well with that in the control subjects, variation of metabolic clearance rate of endogenous C-peptide among the subjects may be negligible for this study. This study provides the precise time course of first- and second-phase insulin response to glucose injection in nonobese and obese subjects with IGT or NIDDM as well as convincing evidence of the progressive reduction of first-phase insulin response with increasing severity of glucose intolerance. First-phase insulin response to glucose might be slightly delayed in some obese subjects with NIDDM.

Analysis of a low-aberration holographic scanner.

Low-aberration holographic scanners that eliminate the need tor lenses or mirrors promise to greatly reduce the cost of laser printers and image scanners. This paper describes how the spot profile of such a scanner can be predicted using the Fresnel-Kirchhoff diffraction integral, and the diffraction efficiency of the scanner can be predicted using Kogelnik's coupled-wave theory. Experimental results verity the accuracy of these design methods. For a prototype scanner used in a high-resolution He-Ne laser printer, the measured linearity error was under +/- 100 microm, and the spot size (half-intensity beamwidth) was under 60 microm for a span (scan width) of 280 mm.

Abnormal calcium handling by perifused pancreatic islets from neonatal streptozotocin diabetic model rats.

To elucidate the mechanism of impaired insulin release in case of non-insulin-dependent diabetes (NIDDM), we investigated insulin release and 45Ca++ efflux from perifused islets obtained from neonatal streptozotocin diabetic model rats. The model rats were prepared by the intraperitoneal administration of 65 mg/kg streptozotocin (STZ) to neonatal males. Rats treated with STZ did not differ from controls in body weight from 1 week to 16 weeks. The model rats had significant hyperglycemia both in the fasting state and after intraperitoneal administration of 2 g/kg glucose. Although the diameter of the islets from the model rats was not significantly different from that of controls, immunoreactivity to anti-insulin was slightly diminished, and degranulation was slightly observed in B-cells. Insulin content was reduced to 45.6% of the control. Insulin release from the perifused islets of STZ-treated rats responded little to 16.7 mmol/L glucose, but normally to 20 mmol/L arginine in the presence of 5.5 mmol/L glucose. In experiments to test the 45Ca++ efflux from the perifused islets prelabeled with 45Ca++, a rise of 45Ca++ efflux concomitant with the second phase of insulin release from the islets of the model rats was inhibited although a sharp increase of 45Ca++ efflux concomitant with the first phase of insulin release was maintained. 45Ca++ uptake for 30 minutes was reduced in the islets from the model rats in the basal and stimulated state of insulin secretion although the incremental 45Ca++ uptake was similar. It is possible that the abnormal calcium handling in pancreatic B-cells may be one of the causes of defect in insulin release in our model rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal pulse sequences for magnetic resonance imaging-computing accurate t1, t2, and proton density images.

Proton nuclear magnetic resonance images (proton MRI) are functions of not only proton density (rho), spin lattice relaxation time (T1), and spin-spin relaxation time (T2) but also MRI scan parameters, so the images differ for different pulse sequences and scan parameters. Systematic measuring errors also vary depending on the type of pulse sequence and the scan parameters. "Pure," objective, T1, T2, and proton density images can be computed from several different Proton MRI, however, systematic measuring errors can cause large deviations between "pure" images obtained using different methods. We tested several scan sequences, and investigated methods to improve the resolution and reduce the errors over the range of T1, T2, and proton density values encountered in representative human tissues. We found that, for given scan time, the combination of inversion recovery 3 spin echo (IR3SE) and saturation recovery 4 spin echo (SR4SE) sequences gave more accurate computed images than other comparable methods tested. It follows, then, that adopting such a sequence as "standard" allows meaningful comparison of clinical results obtained by different researchers.

A new pulse sequence for "fast recovery" fast-scan NMR imaging.

This paper describes the "fast recovery" (FR) method for fast NMR imaging. The FR method combines a sequence of four RF pulses-alternating selective 90 degrees nutation pulses and nonselective 180 degrees pulses-with a gradient field pulse sequence which includes "spoiler" pulses to destroy the coherence between successive sequence cycles. We use the 2-D backprojection method of image reconstruction, but other imaging methods could be applied. The paper analyzes the behavior of the macroscopic magnetization-compares the FR method with other methods and proposes "figure of merit" expressions for relative signal-to-noise (S/N) ratios, scan time reduction ratios, and image contrast-and presents experimental results, including backprojection image reconstruction 2-D images and computed T1 and T2 images. For the FR method, in theory and practice, we find that, after each scan sequence cycle, magnetization is restored to equilibrium quickly and exactly; scan time can consequently be less than a tenth that for the saturation recovery method without any penalty in signal-to-noise ratio. Image contrast is even higher than that of the SR method, and compromise "optimum" sequence (interpulse timing) parameters give high image contrast for a wide range of tissue T1 and T2 (spin-lattice and spin-spin relaxation time) values.

Separation of Plasmodium berghei-parasitized rat erythrocytes by means of carrier-free electrophoresis.

A carrier-free electrophoresis apparatus was used to isolate rat erythrocytes parasitized with Plasmodium berghei. The region of high electrophoretic mobility yielded noninfected erythrocytes, whereas that of low electrophoretic mobility yielded erythrocytes infected with viable parasites. Over 98% purity of parasitized erythrocytes was obtained when blood at an advanced stage of parasitaemia was used. Merozoite-rich fractions were also observed. This continuous one-step separation method should provide large numbers of parasitized erythrocytes for use in immunological and biochemical studies of malaria.