[Quantitative evaluation for scatter and attenuation correction in 99mTc SPECT--multicenter cooperation phantom study].

A multicenter cooperation phantom study was conducted to evaluate the accuracy of a triple energy window scatter correction technique in combination with various attenuation correction methods for 99mTc single photon emission computed tomography (SPECT) imaging. Six centers participated in this research and the data obtained with seven SPECT instruments were analyzed. The phantom used in the experiment was a 20 x 10 cm cylinder filled with homogeneous 99mTc solution, containing two kinds of cold spots (cold rod phantoms). One had a water-filled cylinder 5.5 cm in diameter positioned 2.5 cm from the center. The other contained 6 water-filled cylinders of various sizes. Contrasts of cold regions were in the range from 74% to 120% (true 100%). Another phantom had the shape of a pie-chart divided into six chambers symmetrically positioned in a cylinder 20 cm in diameter and 10 cm in height. Each chamber had volume of 480 ml and contained homogeneous 99mTc solution of different concentrations. This phantom was used to test for linearity between the radio activity concentration and reconstructed count density (linearity phantom). The intercept of the regression line obtained from the linearity phantom was 8.4 kBq ml-1 without scatter correction and -6.8 kBq ml-1 with scatter correction. Contrast was in the range from 78% to 132% (true 100%). The mean relative error for the measured activity concentration was 4.9% +/- 3.5% (mean +/- sd).

How gravity acts on Paramecium: new insights from free-fall experiments.

The swimming behaviour of Paramecium is affected by media of various specific gravities. At 1g, the negative gravitaxis of Paramecium virtually disappears in solutions the specific gravity of which is about the same as that of the organism (1.04). In solutions with a higher specific gravity (1.08), Paramecium becomes positively gravitactic. We recorded the swimming tracks of Paramecium in these media on videotape before, during and after free-falls. The records show that the density-dependent differences in the swimming behaviour disappeared immediately following the onset of the free-fall. The recorded tracks and distributions of cells in the experimental chambers were compared with computer-simulated traces and distributions based on gravitactic and gravikinetic models proposed for Paramecium. Our preliminary analysis favors a novel gravitactic mechanism involving modification of the ciliary movement The drop shaft at the Japan Microgravity Center, Hokkaido (JAMIC) was used for the free-fall experiments.

Relaxation and activation of graviresponses in Paramecium caudatum.

The kinetics of gravitaxis and gravikinesis in Paramecium caudatum were investigated by employing (1) step transitions from normal gravity (1 g) to weightlessness (microgravity) and (2) turns of the experimental chambers from the horizontal to the vertical position at 1 g. The transition to microgravity left existing cell orientations unchanged. Relaxation of negative gravitaxis under microgravity took longer than 10 s and may be described by the time constant of the decay of orientation coefficients. Gravitaxis was started at 1 g by turning the experimental chamber from a horizontal to a vertical position. Gravitaxis activated rapidly during the turning procedure and relaxed to an intermediate level after the turning had stopped. Gravity-induced regulation of swimming speed (gravikinesis) at 1 g had reached a steady state after 1 min; at this point, gravikinesis counteracted the effects of sedimentation (negative gravikinesis). A step transition to microgravity initially reversed the sign of the gravikinesis (positive gravikinesis). The relaxation of this kinetic response was not completed during 10 s of microgravity. The data suggest that gravikinesis is functionally unrelated to gravitaxis and is strongly affected by the rate of change in acceleration. We present a model explaining why gravikinesis reverses sign upon the onset of a step from 1 g to microgravity.

Complete determination of disulfide forms of purified recombinant human serum albumin, secreted by the yeast Pichia pastoris.

In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute. Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable. In this context, disulfide linkages play a significant structural role and their determination is of paramount importance. As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest. The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing. Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined. The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.

Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney.

There are currently no effective therapies for progressive fibrotic diseases. Recent evidence has implicated overproduction of transforming growth factor-beta1 (TGF-beta1) as a major cause of tissue fibrosis. Furthermore, this evidence implies that inhibitors of TGF-beta1 may be clinically useful as antifibrotic agents. The proteoglycan decorin is a known inhibitor of TGF-beta1. In a rat model of glomerulonephritis we have shown that fibrosis is mediated by TGF-beta1. We report here that transfer of decorin cDNA into rat skeletal muscle increases the amount of decorin messenger RNA and protein present in skeletal muscle and decorin present in kidney, where it has a marked therapeutic effect on fibrosis induced by glomerulonephritis. Transfected glomerulonephritic rats showed a significant reduction in levels of glomerular TGF-beta1 mRNA and TGF-beta1 protein, extracellular matrix accumulation and proteinuria. These results demonstrate the potential of gene therapy as a novel treatment for fibrotic diseases caused by TGF-beta1.

Elucidation of the thermal stability of the neutral proteinase II from Aspergillus oryzae.

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.

[Detection of human parvovirus B19 DNA using PCR and serological test using ELISA for diagnosis of infection].

The development of a polymerase chain reaction (PCR) method for detection of human parvovirus B19 DNA and serological test of clinical specimens using ELISA is described. Of 43 serum (or plasma) samples, 29 were found to be positive for B19 DNA using the PCR, anti-IgM was detected in 16 specimens and anti-IgG in 25. In all specimens confirmed to contain B19-specific IgM, the presence of B19 DNA was demonstrated. Furthermore, B19 DNA was demonstrated in a patient with B19 infection for a longer period of time than was anti-IgM. In addition, we discuss the diagnosis of B19 infection using anti-IgG, in the acute stage and convalescent stage. In fetal infection, the PCR method provides sufficient detection of B19 DNA in cord blood, amniotic fluid, ascitic fluid, and fetal pericardial effusion and pleural effusion.

[Left ventricular pressure-volume diagram determined by forward and backward formatting of radionuclide ventriculography and analog pressure data].

Pressure-volume (PV) loop is of great value for the assessment of left ventricular (LV) function, but its clinical application has been limited by methodological complexity. A new system was developed to make accurate loop with simplified procedure, and was applied to clinical and interventional study. The system constitutes of a mobile gamma camera, a poly-amplifier and a data processor (GMS-550U, Toshiba Medical) installed in cardiac catheterization labo for simultaneous raw data handling and successive analysis. Since LV time activity curve (TAC) obtained by usual ECG gating is not fully reliable for a entire cardiac cycle, radionuclide data acquired in list mode was formatted forward and backward from ECG trigger together with analog data of LV pressure, ECG and PCG. PV loops were drawn in 10 patients (OMI, AP, MR, HCM) and 5 normals before and after infusion of angiotensin-II (AII), and Emax and LV work (systolic; SW, diastolic; DW, net; NW = SW - DW) were measured. Radionuclide ventriculography was safely performed with cardiac catheterization even in patients with congestive heart failure. Satisfactory PV loops were obtained by the advantage of simultaneous acquisition of RNV and analog data. Changes of ECG, PCG, volume, pressure and derived indices through one cardiac cycle were readily comparable each other. Peak LV pressure (mmHg) increased from 134 to 159 and then 182 by infusion of AII, but no change in heart rate was observed Emax was higher in normals and AP (mean 1.96 mmHg/ml/m2) than in OMI and MR (range of 0.85-1.36). SW increased in response to rise of LV pressure in all subjects. NW increased in normals and AP, but decreased in OMI and MR with relative increase in DW. In conclusion, this new system is feasible for repetitive studies under drug intervention, since it makes accurate PV loop under physiologic state, i.e. without pacing and volume overloading. Variable changes of SW, DW, and NW in response to afterloading were clarified, which may be useful for the evaluation of cardiac reserve in normal and diseased heart.

Promoting effect of the new chymotrypsin inhibitor FK-448 on the intestinal absorption of insulin in rats and dogs.

FK-448 is a potent and specific inhibitor of chymotrypsin, which enhances the intestinal absorption of insulin in rats and dogs resulting in a decrease in blood glucose levels in these animals. In dogs, the immunoreactive insulin (IRI) level of plasma rose proportionally to the decrease in blood glucose level. From in-vitro data, insulin was inactivated by pancreatic enzymes or the supernatants of intestine or liver homogenates. FK-448 suppressed the digestion of insulin by pancreatic enzymes and its enhancement of the intestinal absorption of insulin was found to be related to its inhibition of digestive enzymes, especially chymotrypsin.

New synthetic inhibitors of chymotrypsin.

4-Substituted carbonylphenyl ester derivatives were prepared and their inhibitory effects on chymotrypsin, trypsin, thrombin, plasmin, pancreatic kallikrein, and plasma kallikrein were examined. Among the various inhibitors tested, 4-[2-(dimethylamino)ethylaminocarbonyl]phenyl 1,2,3,4-tetrahydro-1-naphthoate hydrochloride (FK-316), 4-[(2-[4-pyrrolidinocarbonylmethyl)oxycarbonyl]-phenyl 5-methoxyindole-3-acetate dihydrochloride (FK-375), 4-[(2-[4-(piperidinocarbonylmethylpiperadino]ethyl)oxycarbonyl+ ++]phenyl 1-naphthylacetate dihydrochloride (FK-386), 4-[(2-[4-(2-[morpholino]ethyl)piperadino]ethyl)oxycarbonyl]p henyl 5-methoxy-2-methylindole-3-acetate trihydrochloride (FK-401) and 4-(4-isopropylpiperadinocarbonyl)phenyl 1,2,3,4-tetrahydro-1-naphthoate methanesulfonate (FK-448) were the most effective and specific inhibitors of chymotrypsin.