Focusing and spin polarization of atomic hydrogen beam.

We have developed a spin-polarized-hydrogen beam with a hexapole magnet. By combining the beam chopper and pulsed laser ionization detection, the time-of-flight of the hydrogen beam was measured, and the dependence of the beam profile on the velocity was acquired, which was consistent with the beam trajectory simulations. The spin polarization of the beam was analyzed by using the Stern-Gerlach-type magnet in combination with the spatial scan of the detection laser. The spin polarization was about 95% at a focusing condition due to the hexapole magnet. The polarization was, on the other hand, reduced to about 70% for the beam at higher velocities, which is consistent with simulation results.

Gastric emptying rate in subjects with malocclusion examined by [(13) C] breath test.

Masticatory function is significantly lower in individuals with malocclusion than in those with normal occlusion. Although several studies suggest that masticatory function influences gastrointestinal digestive function, the relationship between malocclusion and gastrointestinal symptoms has not been studied extensively. We hypothesised that insufficient masticatory function would increase the functional burden of the stomach and have some influence on the gastrointestinal system. The purpose of this study was to investigate masticatory function and gastric emptying rate in subjects with malocclusion. Eleven healthy dentate female volunteers and eleven female patients with maloc-clusion underwent a (13) C-acetate breath test with a liquid meal. Maximum (13) CO2 exhalation time (Tmax ) was compared statistically between both groups. Masticatory function was assessed by colour-changeable chewing gum. In addition, the frequency scale for the symptoms of gastroeso-phageal reflux disease (FSSG) and questionnaires on food intake were given to both groups. The mean Tmax of the malocclusion group was significantly longer than that of the normal occlusion group (P = 0·007). Masticatory performance, measured by colour-changeable gum and questionnaires, was significantly lower in the malocclusion group than in the normal occlusion group (P = 0·023, P = 0·003). There was no significant difference in the FSSG results between the two groups (P = 0·262). This study suggested that there was a correlation between malocclusion and gastric emptying function in women.

Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source.

Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of ß-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein.

Postoperative stability of conventional bimaxillary surgery compared with maxillary impaction surgery with mandibular autorotation for patients with skeletal class II retrognathia.

We aimed to compare the postoperative stability of conventional bimaxillary surgery (with bilateral sagittal split osteotomy) with that of maxillary impaction surgery (with mandibular autorotation without bilateral sagittal split osteotomy) in patients with skeletal class II retrognathia. Patients were assigned to have conventional bimaxillary surgery (conventional group, n=6) or mandibular autorotation (experimental group, n=7). Measurements were made using serial lateral cephalometric radiographs taken immediately preoperatively (T0), immediately postoperatively (T1), and one year later (T2) to assess the variation in operative change (T1-T0) and relapse (T2-T1). There was no significant difference in median (range) surgical change in the anterior movement at point B (conventional group, 4.5 (3.0-11.0) mm; experimental group 4.1 (2.1-6.4) mm). However, there was a significant difference in median (range) surgical posterior movement relapse at point B (conventional group -1.7 (-2.3 to -0.5) mm; experimental group -0.6 (-1.0 to 1.0) mm; p=0.032). Mandibular advancement with mandibular autorotation is therefore a more stable procedure than mandibular advancement with bilateral sagittal split osteotomy in patients with skeletal class II retrognathia.

Systemic delivery of IL-10 by an AAV vector prevents vascular remodeling and end-organ damage in stroke-prone spontaneously hypertensive rat.

Interleukin-10 (IL-10) ameliorates various T-helper type 1 cell-mediated chronic inflammatory diseases. Although the therapeutic benefits of IL-10 include antiatherosclerotic effects, pathophysiological effects of IL-10 on vascular remodeling in hypertension have not yet been elucidated. These studies were designed to determine whether sustained IL-10 expression, mediated by an adeno-associated virus (AAV) vector, prevents vascular remodeling and target-organ damage in the stroke-prone spontaneously hypertensive rat (SHR-SP)-an animal model of malignant hypertension. A single intramuscular injection of an AAV1 vector encoding rat IL-10 introduced long-term IL-10 expression. These IL-10-transduced rats had decreased stroke episodes and proteinuria, resulting in improved survival. Histological examination revealed a reduced level of deleterious vascular remodeling of resistance vessels in the brain and kidney of these rats. Immunohistochemical analysis indicated that IL-10 inhibited the enhanced renal transforming growth factor-beta expression and perivascular infiltration of monocytes/macrophages and nuclear factor-kappaB-positive cells normally observed in the SHR-SP. Four weeks after IL-10 vector injection, systolic blood pressure significantly decreased and this effect persisted for several months. Overall, AAV vector-mediated systemic IL-10 expression prevented vascular remodeling and inflammatory lesions of target organs in the SHR-SP. This approach provides significant insights into the prevention strategy of disease onset with unknown genetic predisposition or intractable polygenic disorders.

Ablation of a peptidyl prolyl isomerase Pin1 from p53-null mice accelerated thymic hyperplasia by increasing the level of the intracellular form of Notch1.

Tumor suppressor p53 is essential for checkpoint control in response to a variety of genotoxic stresses. DNA damage leads to phosphorylation on the Ser/Thr-Pro motifs of p53, which facilitates interaction with Pin1, a pSer/pThr-Pro-specific peptidyl prolyl isomerase. Pin1 is required for the timely activation of p53, resulting in apoptosis or cell cycle arrest. To investigate the physiological relationship between Pin1 and p53, we created Pin1-/-p53-/- mice. These p53-deficient mice spontaneously developed lymphomas, mainly of thymic origin, as well as generalized lymphoma infiltration into other organs, including the liver, kidneys and lungs. Ablation of Pin1, in addition to p53, accelerated the thymic hyperplasia, but the thymocytes in these Pin1-/-p53-/- mice did not infiltrate other organs. The thymocytes in 12-week-old Pin1-/-p53-/- mice were CD4(-)CD8(-) (double negative) and had significantly higher levels of the intracellular form of Notch1 (NIC) than the thymocytes of p53-/- or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full-length Notch1 was increased in the thymocytes of Pin1-/-p53-/- mice. Pin1 depletion also inhibited the degradation of NIC by proteasomes. These results suggest that both Pin1 and p53 control the normal proliferation and differentiation of thymocytes by regulating the NIC level.

Examination of bovine lactoferrin binding to bifidobacteria.

In the present study, lactoferrin binding to bifidobacteria and detection of lactoferrin-binding protein in membrane fractions of several bifidobacteria have been demonstrated. This is the first report showing the binding of bovine lactoferrin to four Bifidobacterium spp. (B. infantis, B. breve, B. bifidum, B. longum) incubated with biotinylated lactoferrin and fluorescein conjugated-avidin and observed under an inverted confocal laser scanning microscope. Fluorescence staining showed lactoferrin binding at the pole of the bacterial cells. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the membrane fraction of Bifidobacterium spp. by far western blotting technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on the results of this and previously reported studies, we suggest that binding of lactoferrin to Bifidobacterium longum is strain-dependent.

Photoaffinity labeling of thaumatin-binding protein in monkey circumvallate papillae.

Thaumatin I is an intensely sweet-tasting protein. It was photo-crosslinked with taste papillae of crab-eating monkey by using a conjugated photo-affinity reagent [3H]azidobenzoylthaumatin I. Serial sections of SDS-polyacrylamide gel electrophoresis of the 0.1 M sodium phosphate buffer-soluble fraction from taste papillae had a large peak of radioactivity at the Mr region of approx. 70,000; fractions from non-taste papillae did not. Excess unlabeled thaumatin I reduced the photo-crosslinking at the 70 kDa region; acetylated thaumatin I (which is not sweet) did not. The results show that taste papillae of the monkey contain a protein of Mr approx. 50,000, which binds to thaumatin I (Mr 22,209) but not to completely acetylated thaumatin I. The possibility that the thaumatin-binding protein is a sweet receptor protein is discussed.

Binding of [35S]saccharin to a protein fraction of rat tongue epithelia.

The binding of [35S]saccharin to ammonium sulfate fractions from homogenates of rat tongue epithelia was measured by equilibrium dialysis. The 40--60% saturated ammonium sulfate fraction from the buffer-soluble fraction had the highest saccharin-binding activity. Binding of [35S]saccharin to the 40--60% ammonium sulfate fraction was inhibited by unlabeled saccharin sodium salt. The inhibition increased with increasing unlabeled saccharin concentration and was nearly complete above 10 mM. [35S]Saccharin binding to the 40--60% ammonium sulfate fraction extracted from the tongue epithelia was inhibited by glucose, lactose and sucrose, while binding to similar fractions from tongue muscle was not affected by these sugars. The inhibition of binding of labeled saccharin to the epithelial fraction increased with increasing glucose concentrations. About 35% of the binding was inhibited by 1 M glucose. No significant difference in the amount of inhibition was seen among the three sugars at 0.1 M. The 40--60% ammonium sulfate fraction from tongue epithelium devoid of taste buds bound much less [35S]saccharin than did a similar fraction from epithelium with taste buds. Binding of [35S]saccharin by the preparation from epithelium devoid of taste buds was not inhibited by glucose. The results provide evidence that the 40--60% ammonium sulfate fraction from tongue epithelia with taste buds contains a protein which binds saccharin and sugars. We hypothesize that it is a sweet taste receptor protein.

Structural determination of three neutral oligosaccharides in bovine (Holstein-Friesian) colostrum, including the novel trisaccharide; GalNAc alpha 1-3Gal beta 1-4Glc.

Two trisaccharides, and a pentasaccharide were obtained from bovine colostrum. Their chemical structures were determined by using methylation and 13C-NMR analyses as follows: GalNac alpha 1-3Gal beta 1-4Glc, Gal alpha-1-3Gal beta 1-4Glc, GaL beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc. GalNAc alpha 1-3Gal beta 1-4Glc, which was identified in this study, is a novel oligosaccharide from natural sources. Gal alpha 1-3Gal beta 1-4Glc and Gal beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (lacto-N-novopentaose) have been already found in ovine colostrum, and in horse colostrum and marsupial milk, respectively.

The ABC-exporter genes involved in the lipase secretion are clustered with the genes for lipase, alkaline protease, and serine protease homologues in Pseudomonas fluorescens no. 33.

In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR.

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.

A case of myocarditis associated with IDDM.

We report a case of diabetic ketoacidosis (DKA) complicated by acute myocarditis, which was confirmed by cardiac biopsy. A 26-year-old man was hospitalized with severe DKA. On admission, nonspecific ST-T change was noted on the electrocardiogram (ECG). The patient's levels of creatine phosphokinase (CPK) and glutamic oxaloacetic transaminase were slightly elevated, but he did not complain of chest discomfort or symptoms of heart disease. On the first day after admission, ST-T elevation was noted on ECG during treatment of DKA. By cardiac angiography and cardiac biopsy, coronary heart disease was ruled out and postmyocarditic change was histologically confirmed. An episode of upper respiratory viral infection before the onset of acute diabetes suggested that the patient suffered from viral-induced myocarditis and consequent development of IDDM. This possibility was confirmed by the clinical course of ECG change, with elevated CPK and lactate dehydrogenase and a slightly elevated antibody titer for echovirus.

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain.

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M(r) calculated from the deduced aa sequence was 113,383. Bovine F3 had 93, 94 and 77% aa identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two aa residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.

The calcium-binding property of equine lysozyme.

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio-Gel P-4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metallo-protein like alpha-lactalbumin, which is a homologue of hen egg white lysozyme.

Recombinant bovine lactoperoxidase as a tool to study the heme environment in mammalian peroxidases.

The cDNA encoding bovine lactoperoxidase (LPO) has been expressed in CHO cells. The recombinant LPO was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. rLPO exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Engineering of rLPO into a myeloperoxidase (MPO)-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in MPO. However, the resulting bovine LPO mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of MPO, underlining the complex nature of interactions in the heme vicinity.

Spinal cord ischemia and left atrial myxoma.

A 62-year-old man had an acute, transient, flaccid paraplegia. Examination showed a primary cardiac tumor with emboli to major branches of the aorta. A myxoma was removed from the left atrium, and normal function returned. Left atrial myxoma should be suspected as a cause for embolism to the CNS.

Movement disorders of familial neuroacanthocytosis syndrome.

Characteristic movement disorders were observed in two siblings who had neuroacanthocytosis syndrome with normal serum lipoprotein levels. The disorders included orolingual tic-like movements associated with vocalization, biting of the lip and tongue, peculiar dysphagia with bird-like drinking, and postural lapse with abrupt buckling of the knees. In addition, subtle features of parkinsonism and chorea were observed. These movement problems are strikingly similar to those described in cases of neuroacanthocytosis syndrome in several other familial and sporadic cases. Careful observations of these unusual movement disorders may provide a clue to the diagnosis of this rare syndrome.

A case of familial amyloid polyneuropathy homozygous for the transthyretin Val30Met gene with motor-dominant sensorimotor polyneuropathy and unusual sural nerve pathological findings.

OBJECTIVE: To report a case of familial amyloid polyneuropathy homozygous for the amyloidogenic transthyretin (ATTR) Val30Met gene with motor-dominant sensorimotor polyneuropathy and unusual sural nerve pathological findings. METHODS: Mass spectrometry analysis and polymerase chain reaction-restricting fragment length polymorphism were performed. A right sural nerve biopsy specimen was obtained for histological investigation. SETTING: Academic medical center. RESULTS: A 56-year-old Japanese man living in a local town (Nakajima, Japan) in Ishikawa Prefecture, a nonendemic area of type I familial amyloidotic polyneuropathy, had vitreous amyloidosis, motor-dominant sensorimotor polyneuropathy, erectile dysfunction, and urinary incontinence. He had neither orthostatic hypotension nor indolent diarrhea. Restriction enzyme analysis with EcoT22 I of amplified DNA and mass spectrometry analysis revealed homozygosity for ATTR Val30Met. Of 8 family members, 5 were evaluated and found to be heterozygous for ATTR Val30Met; a family history found no relative with the similar neurologic disorders. The sural nerve biopsy specimen showed focal edema and an amyloid deposit in the subperineural tissue, associated with moderate loss of myelinated and unmyelinated fibers. CONCLUSIONS: In addition to the findings characteristic of homozygosity for ATTR Val30Met such as vitreous amyloidosis and relatively less autonomic involvements, this case had the unique findings of motor-dominant sensorimotor polyneuropathy and unusual sural nerve biopsy specimen results.

Improved generation of catalytic antibodies by MRL/MPJ-lpr/lpr autoimmune mice.

To compare the abilities of different strains of mice to elicit catalytic antibodies (Abs), we determined the occurrence of esterolytic Abs in BALB/c (normal strain) and MRL/MPJ-lpr/lpr (MRL/lpr, autoimmune) mice after immunization with the transition state analog (TSA) 1. Hybridoma supernatants elicited against TSA 1 were screened by ELISA for binding to the BSA-conjugated TSA 1 (=3b), and then screened for binding to the BSA-linked short TSA 2 (=4). We obtained eight times more positives from MRL/lpr mice than from BALB/c mice by these screening steps. The monoclonal antibodies (mAbs) obtained here were examined for binding and catalytic activity. Fifteen of 25 mAbs from MRL/lpr had esterolytic activity, compared with only two of 21 mAbs from BALB/c. These results demonstrated that the occurrence of catalytic Abs was much higher in MRL/lpr mice than in BALB/c mice, which is in good agreement with the previous report by Tawfik et al. [Tawfik, D.S., Chap, R., Green, B.S., Sela, M., Eshhar, Z., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 2145-2149] using a different kind of TSA. Thus, these studies strongly suggest that using the appropriate strain can be a key factor in the efficient production of catalytic Abs. Furthermore, these mAbs were characterized to elucidate the mechanism of strain difference, and determine whether MRL/lpr mice can be used with other TSAs for the efficient production of catalytic Abs.