Shinichi Kobayashi

Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials

Non-enzymatic proteins were added during hydrolysis of cellulose and simultaneous saccharification and fermentation (SSF) of different biomass materials. Bovine serum albumin (BSA), a model non-enzymatic protein, increased cellulose and xylose conversion efficiency and also enhanced the ethanol yield during SSF of rice straw subjected to varied pretreatments. Corn steep liquor, yeast extract, and peptone also exerted a similar effect as BSA and enhanced the enzymatic hydrolysis of rice straw. Compared to the glucose yields obtained after enzymatic hydrolysis of rice straw in the absence of additives, the glucose yields after 72h of hydrolysis increased by 12.7%, 13.5%, and 13.7% after addition of the corn steep liquor, yeast extract, and peptone, respectively. This study indicated the use of BSA as an alternative to intensive pretreatment of lignocellulosic materials for enhancing enzymatic digestibility. The utilization of non-enzymatic protein additives is promising for application in glucose and ethanol production from lignocellulosic materials.

Phase diagrams and ionic defects in the 2212 and 2201 phases of the bismuth system

Abstract The compositional diagrams representing the stability regions of the 2212 and 2201 phases of the bismuth system were constructed for several temperatures above 820°C. The actual densities of both phases were determined by pycnometry and compared with the theoretical densities. It was found that the maximum ion deficiency is about 2% for the 2212 phase and nearly zero for the 2201 phase. The valences of Bi and Cu were determined by chemical analysis and the excess oxygen was calculated. The maximum excess oxygen was found to be 2.5% and 3% for the 2212 and 2201 phases, respectively. From the ion deficiency and the excess oxygen, it was concluded that the excess oxygen occupies not the vacancy but the interstitial site.

Effect of Bovine Serum Albumin (BSA) on Enzymatic Cellulose Hydrolysis

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48xa0h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3xa0mg/g substrate, each with different initial BSA concentration treatments at 50xa0°C; in addition, more cellulase was adsorbed onto the filter paper at 50xa0°C compared with 35xa0°C. After 48xa0h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41xa0% when treated with 1.0xa0mg/mL BSA. Even after 96xa0h of hydrolysis, 25xa0% still remained. Meanwhile, after 48xa0h of incubation without substrate, the remaining enzyme activities were increased 20.7xa0% (from 43.7 to 52.7xa0%) and 94.8xa0% (from 23.3 to 45.5xa0%) at 35 and 50xa0°C, respectively. Moreover, the effect of the BSA was more obvious at 35xa0°C compared with 50xa0°C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50xa0°C, the cellulose conversion was increased from 75xa0% (without BSA treatment) to ≥90xa0% when using BSA dosages between 0.1 and 1.5xa0mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

Advance in animal experiments with the undulation pump total artificial heart: 50 and 54 day survival periods with 1/R control

The undulation pump total artificial heart (UPTAH) is a unique, implantable, total artificial heart (TAH) that uses undulation pumps. To achieve long-term survival in animals with physiologic hemodynamic conditions, a control method based on conductance and arterial pressure was applied to UPTAH. With this control method, called 1/R control, survival periods of 50 days (No. 0016, 49.6 kg) and 54 days (No. 0030, 42.5 kg) were obtained in adult female goats. In No. 0016, 1/R control was applied to the left pump, whereas in No. 0030, it was applied to the right pump. Another pump was used for left-right balance control. The control stability was better in No. 0030 than in No. 0016. The sucking effect of the left atrium was remarkable in No. 0016, possibly because of a time delay when left-right balance control was performed with the right pump. In No. 0016, the cause of death was probably a thrombus flown from a panus in the left atrium. It is possible that the left atrial suction effect influenced the thrombus and panus formation in the left atrium. In No. 0030, the cause of death was a small rupture of the membrane in the right pump. The rupture may have been caused by excessive negative pressure inside the pump. This pressure resulted from suction of the right atrium because of an unexpected control excursion, which was probably caused by a software bug. It will be necessary to redesign the undulation pump and improve the software to achieve longer survival periods for animals with physiologic hemodynamic conditions.

The Effect of Nonenzymatic Protein on Lignocellulose Enzymatic Hydrolysis and Simultaneous Saccharification and Fermentation

Nonenzymatic protein was added to cellulase hydrolysis and simultaneous saccharification and fermentation (SSF) of different biomass materials. Adding bovine serum albumin (BSA) and corn steep before cellulase enhanced enzyme activity in solution and increased cellulose and xylose conversion rates. The cellulose conversion rate of filter paper hydrolysis was increased by 32.5xa0% with BSA treatment. When BSA was added before cellulase, the remaining activity in the solution was higher than that in a control without BSA pretreatment. During SSF with pretreated rice straw as the substrate, adding 1.0xa0mg/mL BSA increased the ethanol yield by 13.6xa0% and final xylose yield by 42.6xa0%. The results indicated that lignin interaction is not the only mechanism responsible for the positive BSA effect. BSA had a stabilizing effect on cellulase and relieved cumulative sugar inhibition of enzymatic hydrolysis of biomass materials. Thus, nonenzymatic protein addition represents a promising strategy in the biorefining of lignocellulose materials.

The p-Adic Height Pairing on Abelian Varieties at Non-ordinary Primes

In P. Schneider (Invent Math 69(3):401–409, 1982) constructed the p-adic height pairing on abelian varieties at ordinary primes by using the universal norm subgroup. In this paper, we generalize his construction to the non-ordinary case and compare it with that of Zarhin-Nekovar. As an application, we generalize the p-adic Gross-Zagier formula in Kobayashi (Invent Math 191(3):527–629, 2013) to newforms for Γ0(N) of weight 2 with arbitrary Fourier coefficients (not necessarily in (mathbb{Q})).

p-ADIC EISENSTEIN-KRONECKER SERIES FOR CM ELLIPTIC CURVES AND THE KRONECKER LIMIT FORMULAS

Consider an elliptic curve defined over an imaginary quadratic field K with good reduction at the primes above p ≥ 5 and with complex multiplication by the full ring of integers of K. In this paper, we construct p -adic analogues of the Eisenstein-Kronecker series for such an elliptic curve as Coleman functions on the elliptic curve. We then prove p -adic analogues of the first and second Kronecker limit formulas by using the distribution relation of the Kronecker theta function.

Quantification of PrPC in bovine peripheral tissues: Analysis in wild-type and PrPC-deficient cattle

Cellular PrP (PrPC) is necessary for bovine spongiform encephalopathy (BSE) infection. The purpose of the present experiment was the quantification of PrPC in peripheral tissues to assess the risk of BSE infection from these tissues. The tissue distribution of PrPC was examined by a sandwich enzyme-linked immunosorbent assay (sELISA) and histochemical analysis. PrPC-deficient cows were used as a negative control. The sELISA revealed that the brain contained the highest PrPC content (10.7 µg/g tissue), while other organs/tissues harbored lower amounts, in decreasing order as follows: longissimus capitis muscle, iliocostalis thoracis muscle, splenius muscle, biceps femoris muscle, triceps brachii muscle, longissimus thoracis muscle, ileum, jejunum, duodenum, colon, cecum, apex linguae, omotransversarius muscle, posterior part of the corpus linguae, anterior part of the corpus linguae and radix linguae (5.2- to 31-fold less PrPC than the brain). In the tissue/organs of PrP-deficient cows, PrPC levels were under the limit of detection. Histochemical analysis showed that PrPC was expressed in nerve cells in intestinal tissues. The presence of PrPC in the bovine tongue, skeletal muscles and intestines raises the possibility of PrPSc accumulation in these tissues, indicating that these organs/tissues may serve as potential sources of BSE infection.

Abstract LB-172: A phase I clinical trial of RNF43 peptide-specific immune cell therapy combined with low-dose cyclophosphamide for patients with advanced solid tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnAccumulated clinical studies have validated the efficacy of immunotherapies for patients with solid tumors. Although effective anti-tumor immune responses have been introduced in many clinical trial cases, several immunosuppressive factors such as regulatory T cells (Tregs) have been considered to inhibit induction of anti-tumor immunity.nnWe have developed new approach of immunotherapy in order to augment anti-tumor effect by expanding tumor-specific cytotoxic T cells (CTL), and eliminate Tregs by utilizing chemotherapy. We have recently reported the efficacy of multiplex vaccination with HLA-A*2402 restricted tumor-associated antigens peptides (TAA), preceded by the administration of low-dose cyclophosphamide (CPM) in order to eliminate Tregs. This time, we have been conducting a phase I clinical trial to study the utilization of CTL after CPM administration followed by a consequent administration of dendritic cells (DC) and IL-2. This study targets the novel HLA-A*0201 or HLA-A*2402-restricted TAA, RING finger protein 43 (RNF43), and is designed as a dose-escalation study of CTL. The purpose of this trial is to evaluate the safety and tolerability of the proposed method as primary endpoint and the efficacy including over all survival and immune responses as secondary endpoint.nn11 patients with advanced solid tumors refractory to standard therapy were enrolled in this trial. They were HLA-A*2402 or A*0201 positive and exhibiting RNF43 expression levels in their tumor cells more than those in RNF43-positive control cell (HCT15). Primarily, severe adverse event greater than Grade 3 was not observed in our patients. Clinical responses of stable disease (SD) were observed in 8 out of 11 patients. Among them, 2 patients showed a decrease in the tumor markers with stabilized tumor sizes during the observed period, and 1 patient showed a mixed response. On the other hand, 3 other patients showed progressive disease (PD). The number of Tregs significantly decreased after the administration of CPM (p=0.033), and the higher decreasing rate of Tregs was related to the good clinical response. In CD107a/b mobilization assay, the ratio of RNF43-specific CD8 T cells in SD increased with time, conversely that in PD decreased (p=0.005). Also, in Intracellular Staining Assay, the ratio of IFN-g produced by RNF43-specific CD8 T cells was significantly correlated with clinical benefit (p=0.02), while TNF-α and IL-2 were not. Consequently, the combination of immunotherapy and CPM may induce tumor specific immune cells accompanied by the decreased number of Tregs.nnIn conclusion, this trial is tolerable and may be clinically efficient against advanced solid tumors. Further immunological assessment should be conducted in order to explore the valid biomarkers to predict clinical efficacy before treatment.nnCitation Format: Yasuki Hijikata, Toshihiko Okazaki, Hiroyuki Inoue, Yoshihiro Tanaka, Kenzaburo Tani, Shinichi Kobayashi, koji Yoshida. A phase I clinical trial of RNF43 peptide-specific immune cell therapy combined with low-dose cyclophosphamide for patients with advanced solid tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2014-LB-172

The Local Root Number of Elliptic Curves

In this paper, we compute the sign of the functional equation of the L-function of elliptic curves in terms of the coefficients of the Weierstras equation.