Enzyme immobilization by radiation-induced polymerization of hydrophobic glass-forming monomers at low temperatures.

Enzyme immobilization was studied by means of radiation-induced polymerization of hydrophobic glass-forming monomers at low temperatures. The polymerized hydrophobic composite was generally obtained in microspheric form. Enzymatic activity showed little decrease with repeated use in these systems. The particle size of the microsphere increased with increasing monomer concentration, and activity yield had a maximum at an optimum monomer concentration. Immobilization by copolymerization of hydrophilic and hydrophobic comonomers was also investigated and a maximum activity yield was found at a certain monomer concentration. A model scheme for immobilization at low temperatures was proposed and discussed.     

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke)

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b^* values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.     

Properties of thermolysin immobilized in polymer matrix by radiation polymerization

Thermolysin (Bacillus thermoproteolyticus neutral proteinase, EC 3.4.24.4) has been immobilized by radiation polymerization of hydrophilic and hydrophobic monomers, and its properties, such as enzyme activity, thermal stability and durability, have been studied. The activity of the immobilized enzymes increased with an increase in the hydrophilicity of the polymer matrix and with a decrease in monomer concentration. Immobilization with hydrophilic monomers increased the thermal stability of the enzymes, but the thermal stability of the enzymes immobilized with hydrophobic monomers was comparable with that of native enzymes. The durability of the immobilized enzymes was examined by continuous hydrolysis of casein; enzymes immobilized with a high concentration (90%) of hydrophilic monomers appeared to be stabilized and could be used for long times.     

Do highly trained monkeys walk like humans? A kinematic study of bipedal locomotion in bipedally trained Japanese macaques

In this study, we examined the kinematics of bipedal walking in macaque monkeys that have been highly trained to stand and walk bipedally, and compared them to the kinematics of bipedal walking in ordinary macaques. The results revealed that the trained macaques walked with longer and less frequent strides than ordinary subjects. In addition, they appear to have used inverted pendulum mechanics during bipedal walking, which resulted in an efficient exchange of potential and kinetic energy. These gait characteristics resulted from the relatively more extended hindlimb joints of the trained macaques. By contrast, the body of the ordinary macaques translated downward during the single-limb stance phase due to more flexed hindlimb joints. This resulted in almost in-phase fluctuations of potential and kinetic energy, which indicated that energy transformation was less efficient in the ordinary macaques. The findings provide two insights into the early stage of the evolution of human bipedalism. First, the finding that training considerably improved bipedal walking a posteriori may explain why the very first bipeds that might not yet have been morphologically adapted to bipedal walking continued to walk bipedally. The evolutionary transition from quadrupedalism to bipedalism might not be as difficult as has been envisioned. In addition, the finding that macaques, which are phylogenetically distant from humans and in which bipedal walking is unlike human walking, could develop humanlike gait characteristics with training, provides strong support for the commonly held but unproven idea that the characteristics of the human gait are advantageous to human bipedalism.     

Factors influencing eating ability of old in-patients in a rehabilitation hospital in Japan

Objectives: This study was designed to determine the factors influencing eating ability of old in‐patients in a rehabilitation hospital. Design: Cross‐sectional investigation. Setting: Forty‐six in‐patients in the rehabilitation ward of Hashimoto Hospital in Kagawa Prefecture in Japan were investigated using a multi‐disciplinary approach. Main outcome measures: Age, gender, state of dentition, muscle activity of lip, cheek and tongue, biting force, salivary flow rate per a minute (SFR), masticatory ability for gummy jelly, swallowing ability, texture of meal, independency of walking (Functional Independence Measure=FIM) and ability to communicate. Results: Bivariate analysis for the relationship between surveyed items and masticatory ability (chi‐square test) identified that better masticatory ability for gummy jelly was associated with age (<85 years), gender (male), state of dentition (dentate), SFR (high), activity of lip (good), biting force (high), swallowing ability (good) and activity of communication (high). Among these items, SFR (p=0.001), gender (p=0.004), ability to communicate (p=0.005) and age (p=0.012) were found having an influence on the masticatory ability (logistic regression analysis). On the other hand, age (<85years), gender (male), SFR (high), activity of lip (good), activity of cheek (good), biting force (high), masticatory ability (good) and swallowing ability (good) had a relationship with normal texture of meal. In regression analysis, only two items, activity of lip (p=0.003) and swallowing ability (p=0.024) emerged as factors on texture of meal. Conclusions: Masticatory ability for gummy jelly was influenced by cognitive function and was excluded from the factors on the state of meal. These results suggested the limitation of evaluation using test food, so dentists should observe eating behaviour of in‐patients. In addition, dentists should pay attention to the activity of the lip and swallowing ability as well as dentition and prostheses in the rehabilitation of eating ability. As SFR was the most significant factor on masticatory ability, this emphasizes the necessity of care for dry mouth caused by side effects of multi‐medication     

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4alpha causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4alpha in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4alpha protein, due, in part, to the limited availability of human HNF4alpha-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4alpha fusion proteins as the immunizing agent. The mouse anti-human HNF4alpha monoclonal antibody (K9218) generated against human HNF4alpha1/alpha2/alpha3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4alpha proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4alpha protein, but HEK293 showed no expression of HNF4alpha protein. Nuclear-specific localization of the HNF4alpha protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4alpha protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4alpha protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4alpha in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4alpha mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4alpha and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4alpha isoforms in humans and in several other mammalian species.     

Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRalpha) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRalpha protein level in the cell is low and the LXRalpha protein itself is very hard to detect. We have previously reported that the mRNA for LXRalpha is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRalpha protein in the human macrophage, we have established a monoclonal antibody against LXRalpha, K-8607. The binding of mAb K-8607 to the human LXRalpha protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRalpha antibody should prove to be a useful tool in the analysis of the human LXRalpha protein.