Reversible Dissociation of Crystalline Yeast Phosphoglyceric Acid Mutase in Alkali

The structure of crystalline yeast phosphoglyceric acid mutase has been investigated by sedimentation-velocity and equilibrium measurements, optical rotatory dispersion measurements and viscometry. The data indicate that this enzyme is a globular, compact and highly organized protein with a low helix content. The native structure remains unchanged at pH 10.5. Dissociation of the enzyme into subunits has been observed at pH values of 11.5 and above. From optical rotatory dispersion measurements, it is found that the enzyme loses a large part of its organized conformation when it dissociates in alkaline solution. On neutralization, the alkali-treated enzyme regains its activity. The ability to regain the enzyme activity is gradually lowered with the increase of pH value to be incubated and with time of exposure. Inactivation at pH 13.0 is almost irreversible. However, the reversibility of the inactivation at pH 13.0 is appreciably enhanced by the presence of phosphate compounds in the reactivation system. Particulary, it is found that presence of substrates or the coenzyme is effective for considerable improvement of the reversibility. Molecular weight analyses by ultracentrifugation indicate that subunits have approximately equal molecular weights and that the native enzyme is consisted of four polypeptide chains.     

Factors Affecting Developmental Increase of Tyrosine Aminotransferase in Rat Liver Immediately after Birth

Labeling with ³⁵S-methionine of dispersed hepatocytes prepared from neonatal rat liver and successive immunoprecipitation with antiserum against tyrosine aminotransferase (TAT) indicated that increase of TAT activity to a peak about 12 hours after birth and the decrease thereafter are mainly due to changes of TAT synthesis. Similar changes of TAT activity was also observed in the livers of premature neonates which were taken out by Caesarian section and nursed by foster mothers. This indicated that the appearance of TAT activity at this period is not an event programmed along with fetal development but is triggered by birth. The level of glucagon in neonatal plasma increased after birth. Administration of glucagon to neonates caused a great increase of TAT activity whereas the effect of dexamethasone was not so evident. These suggested that glucagon is an important factor affecting the abrupt appearance of TAT after birth.     

MicroRNA profile predicts recurrence after resection in patients with hepatocellular carcinoma within the Milan Criteria

Objective

Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection.

Methods

We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out cross-validation method, and the time-averaged area under the ROC curve (ta-AUROC).

Results

The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P<0.0001). The best ta-AUROC using the whole dataset, T-miRs, N-miRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P<0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the ‘field effect’.

Conclusion

microRNA profiling can predict HCC recurrence in Milan criteria cases.     

Intracellular Localization and Characterization of Beef Liver Aldehyde Dehydrogenase Isozymes

Various physiological roles of mammalian aldehyde dehydrogenase had been anticipated because of its broad substrate specificity. In order to clarify roles of the enzyme and the regulation of aldehyde metabolisms in liver, the intracellular distribution and isozyme of beef liver aldehyde dehydrogenase were studied.

The presence of the mitochondrial, the microsomal and the cytoplasmic isozymes were proved by the isoelectric focusing. These isozymes were different from each other in pH-activity curve in the responces for steroid hormones and disulfiram.

It was suggested by comparing the reactivities of these isozymes for various aldehydes that particular aldehyde might be oxidized by a favorite isozyme at particular locality in the liver cells and that a share of physiological role among these isozymes is probable.     

Studies on Crystalline Yeast Phosphoglycenc Acid Mutase

Effects of pH and some chemical agents (1) on the solubilization of insoluble pectin bound to suspended particles, (2) on the viscosity decrease of native soluble pectin, (3) on the flocculation of suspended particles and (4) on the over-all clarification of apple juice have been studied Optimum pH ranges were: (1), 3.2~4.0; (2), 3.6~4.1; (3), lower than 3.0; (4), 3.2~3.8. Gelatin had no effect on (1) and (2) but stimulated (3) and (4). NaCl had no detectable effect on (2) and (3) but slightly stimulated (1) and (4). CaCl₂ strongly inhibited (1), (2) and (3). SnCl₄ stimulated (3) but strongly inhibited (1), (2) and (4). EDTA (ethylenediamineteraacetic acid) accelerated (1) and (4), and had no detectable effect on (2) and (3).     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Immunohistochemical demonstration of cholinergic structures in central ganglia of the slug (Limax maximus, Limax valentianus)

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans.     

A ganglioside-induced toxic soluble Abeta assembly. Its enhanced formation from Abeta bearing the Arctic mutation

The mechanism underlying plaque-independent neuronal death in Alzheimer disease (AD), which is probably responsible for early cognitive decline in AD patients, remains unclarified. Here, we show that a toxic soluble Abeta assembly (TAbeta) is formed in the presence of liposomes containing GM1 ganglioside more rapidly and to a greater extent from a hereditary variant-type ("Arctic") Abeta than from wild-type Abeta. TAbeta is also formed from soluble Abeta through incubation with natural neuronal membranes prepared from aged mouse brains in a GM1 ganglioside-dependent manner. An oligomer-specific antibody (anti-Oligo) significantly suppresses TAbeta toxicity. Biophysical and structural analyses by atomic force microscopy and size exclusion chromatography revealed that TAbeta is spherical with diameters of 10-20 nm and molecular masses of 200-300 kDa. TAbeta induces neuronal death, which is abrogated by the small interfering RNA-mediated knockdown of nerve growth factor receptors, including TrkA and p75 neurotrophin receptor. Our results suggest that soluble Abeta assemblies, such as TAbeta, can cause plaque-independent neuronal death that favorably occurs in nerve growth factor-dependent neurons in the cholinergic basal forebrain in AD.     

Reduction of Aluminum Toxicity by 2-Isopropylmalic Acid in the Budding Yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae secretes 2-isopropylmalic acid (2-iPMA), an intermediate in leucine biosynthesis. Because 2-iPMA binds Al(III) in the culture medium, it is thought to reduce toxicity by Al(III). The effects of 2-iPMA and malic acid (MA) on Al toxicity were investigated in a medium with a low pH and low concentrations of phosphates and magnesium. The reduction in the growth of S. cerevisiae observed in the presence of 100 μM Al(III) ions was relieved more by the addition of 1.0 mM 2-iPMA than by 1.0 mM MA, indicating that 2-iPMA possesses superior Al(III)-ion detoxification ability. Investigations using the wild type and the Δleu4 and Δleu9 mutant strains indicated that secretion of a sufficient level of 2-iPMA was required to enhance the Al tolerance. It is thought that 2-iPMA secreted from the yeast cells chelates Al ions and prevents them from entering the cells, resulting in Al tolerance. Suzuki and Tamura contributed equally to this work.