Acid-Stable α-Amylase of Black Aspergilli

The acid-stable α-amylase or the acid-unstable α-amylase from Aspergillus niger contained 24 moles or 7 moles mannose and 4 moles or 1 mole hexosamine per mole of protein, respectively.

The acid-stable α-amylase and the acid-unstable α-amylase contained calcium only, but not detectable amounts of other metals. Calcium contents of the both enzymes were converged to at least one gram atom per mole of enzyme by dialysis against acetate buffer. The last calcium could be removed under the suitable conditions by EDTA. Calcium removal by EDTA was accompanied by the loss of activity and by the little change of UV absorption spectra. The phenomenon caused by calcium removal were partially reversible. This last one atom of calcium seemed to be essential for the maintenance of active structure of α-amylase.     

Constant copy numbers of plasmids inSaccharomyces cerevisiae hosts with different ploidies

Summary The cellular -galactosidase activities produced by thelac'Z gene ofEscherichia coli, cloned on YEp, YRp, or YCp-type plasmids in host cells ofSaccharomyces cerevisiae with different ploidies, and which was expressed by a modified jeastHIS5 promoter, showed characteristic differences depending on the plasmid. But for any given plasmid, the isogenic diploid and tetraploid transformants showed slightly lower enzyme activities than their respective haploid transformants. This was due to the similar copy numbers of the plasmids in host cells. Since the cell number per unit volume of the culture decreased with increasing cell ploidy, the enzyme activity per unit volume of the culture decreased significantly. The holding stability of plasmids increased with increasing ploidy of the host cell, especially that of the YRp plasmid. On the YRp plasmid, thelac'Z gene showed higher productivity withTRP1 thanLEU2 as the selection marker for the plasmid.     

Tissue-specific interactions of TNI isoforms with other TN subunits and tropomyosins in C. elegans: the role of the C- and N-terminal extensions

The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.     

Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies

Abstract We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyl-transferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans . The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.     

The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.     

In vivo singlet-oxygen generation in blood of chromium(VI)-treated mice

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).     

Rapid and Dense Culture of Acinetobacter calcoaceticus on Palm Oil

A soil microorganism, identified as Acinetobacter calcoaceticus KB-2, was cultivated on palm oil as a carbon source for cell production. This organism grew with a specific growth rate of l.lOh^?1. The pH optimum for growth was between 6.5 and 7.0, and the temperature optimum was 39°C. Compared with other strains on water-insoluble substrates such as hydrocarbons and natural oils and fats so far reported, the cultivation time for this strain was short and the cell mass productivity was relatively high. More than 90% of the palm oil was assimilated by this strain, and the overall cell yield was 1.02 (g of cells/g of palm oil) after 8 hr cultivation with the concentration of 3% palm oil.     

Glutamic acid potentiates hepatocyte response to mitogens in primary culture

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L-15 medium. Addition of glutamic acid (1–20 μM) to EGF-containing L-15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF-induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentrations of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose-dependent (0.5–20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5–20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [¹²⁵I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens © 1994 Wiley-Liss, Inc.