Production of l-alanine from ammonium fumarate using two immobilized microorganisms

Summary For the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate -decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.     

Involvement of τ Protein Kinase I in Paired Helical Filament-Like Phosphorylation of the Juvenile τ in Rat Brain

Abstract: τ protein kinase I (TPKI) phosphorylates τ and forms paired helical filament epitopes in vitro. We studied temporal expression and histochemical distribution of τ phosphoserine epitopes at sites known to be phosphorylated by TPKI. Antibodies directed against phosphorylated Ser¹⁹⁹ (anti-PS 199) or phosphorylated Ser³⁹⁶ (C5 or anti-PS 396) were used. TPKI is abundantly expressed in the young rat brain and the highly phosphorylated juvenile form of τ occurs in the same period. The activity peak of TPKI coincided with the high level of phosphorylation of Ser¹⁹⁹ and Ser³⁹⁶ in juvenile τ at around postnatal day 8. By immunohistochemistry on the hippocampus and neocortex of 3–11-day-old rats, phosphorylated Ser³⁹⁶ was found in young axonal tracts and neuropil, where TPKI immunoreactivity was also detected. TPKI and phospho-Ser¹⁹⁹ immunoreactivities were also detected in the perikarya of pyramidal neurons. TPKI immunoreactivity had declined to a low level and phosphorylated serine immunoreactivities were undetectable in the sections of adult brain. These findings implicate TPKI in paired helical filament-like phosphorylation of juvenile form of τ in the developing brain.     

Cloning and analysis of the gene encoding lectin from the acorn barnacle Megabalanus rosa

In the acorn barnacle Megabalanus rosa, two types of galactose-binding C-type lectins (BRA-2 and BRA-3) have been identified. Here, we report the isolation and characterization of BRA-2 cDNA and genomic clones. In contrast to the BRA-3 gene, which consists of four exons, BRA-2 is encoded by a single exon, implying differences in the physiological roles of the two lectins.     

Identification of a Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein Which Is Recognized by a Monoclonal Antibody 5-2-26

We have investigated a phosphatase-sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5-2-26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of ³²P-labeled N protein showed that the protein contained phosphoserine and phospho-threonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase-sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino-acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino-acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354–389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine-389 by threonine also reduced the antigenicity. These results strongly suggest that serine-389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5-2-26.     

High temporal resolution video imaging of intracellular calcium

We have developed a system for imaging intracellular free calcium ion concentration ([Ca2+]i) at the highest rate possible with conventional video equipment. The system is intended to facilitate quantitative study of rapid changes in [Ca2+]i in cells that move. It utilizes intensified video cameras with nearly ideal properties and digital image processing to produce two images that can be ratioed without artifacts. Two dichroic mirrors direct images of cellular Indo-1 fluorescence at two different wavelengths to two synchronized video cameras, each consisting of a fast micro-channel plate image intensifier optically coupled with a tapered fiber optic bundle to a CCD image sensor. The critical technical issues in this dual-image system are: (1) minimization and correction of the small geometric and other types of differences in the images provided by the two cameras; and (2) the signal-to-noise ratio that can be achieved in single frames. We have used this system to obtain images of [Ca2+]i at 16.7 ms intervals in voltage-clamped single cardiac cells perfused internally with Indo-1 (pentapotassium salt). The images indicate that, except for the nuclear regions, [Ca2+]i is uniform during normal excitation-contraction coupling. In contrast, changes in [Ca2+]i propagate in rapid 'waves' during the spontaneous release of Ca2+ that accompanies certain 'Ca2(+)-overload conditions.'     

Profile control of the specific growth rate in fed-batch experiments

Summary The aim of this paper is to apply a computer control scheme to a laboratory scale fermentor so that the specific growth rate in a baker's yeast fed-batch culture, which cannot be measured directly, will follow as accurately as possible the desired profile specified in advance. Using an extended Kalman filter and programmed controller/feedback compensator (PF) system proposed previously, profile control of the specific growth rate () was achieved experimentally in a baker's yeast fed-batch culture. Also, bang-bang type profile control of minimized the proportion of budding cells, which have a strong correlation with the fermentative activity in bread-making.     

LUXURY PHOSPHATE UPTAKE AND VARIATION OF INTRACELLULAR METAL CONCENTRATIONS IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)1

The effectr of phosphate starvation and subsequent uptake on distribution and concentration of phosphate metabolic intermediates and metals were studied in Heterosigma akashiwo (Hada) Hada by ³¹P-NMR spectroscopy, neutron activation analysis and ESR spectroscopy. Excess orthophosphate (4.5 μM P_i, as NaH₂PO₄) added to a medium with P-depleted H. akashiwo cells was rapidly taken up resulting in an increase in P cell quota (q_p)from 68.2 to 99.6 fmol. cell-¹in 2 h and to 156.3 fmol. cell-¹in 6 h. After three days, q_p approached about 190 fmol. cell^?1. Polyphosphate (PP_i) rapidly increased from 0 to 11.4 fmol· cell^?1in 2 h and to 24.7 fmol·cell^?1in 6 h. Diel variation of cell quota indicated that cellular P_i increase was synchronized with cellular PP_i decrease and vice versa. The average chain length of PP_i increased from ca. 0 to ca. 10.2 phosphate residues in 2 h after addition of P_i and one day later, from ca. 9.8 to ca. 12.5. The cell quota of Mn (q_Mn), and to a lesser extent Co, increased rapidly from 4.87 fg. cell^?1in the P- starved condition to 50.48 fg·cell^?12 h afer addition of P_i but decreased to 8.63 fg. Cell^?1by 6 h. Concentrations of Zn, As, Hf, Cu and sometimes Al, Mg, K, and Ca changed in a manner opposite to that of Mn and Co. The excretion of these cations, which was synchronized with the uptake of Mn and Co, may be important for a charge balancing in the cells. The ESR spectra showed that the high cellular Mn observed at 2 h after P addition was Mn²⁺which was taken up by the cells rather than adsorbed on the cell surface. These data combined with PP_i data suggested that the behavior of q_Mn is synchronized with the behavior of average chain length of PP_i.     

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non-Catalytic Subunit (P Protein)

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H]leucine and [³²P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).