Optimization of theaflavin biosynthesis from tea polyphenols using an immobilized enzyme system and response surface methodology

Theaflavins were synthesized from tea polyphenols extracted from green tea using an immobilized polyphenol oxidase system. To optimize the production of theaflavins, response surface methodology was applied to determine the effects of five critical variables and their mutual interactions on theaflavin biosynthesis at five levels. A total of 52 individual experiments were performed and a statistical model predicted that the highest theaflavin concentration was 0.766mgml^–1 at optimized conditions. Using these optimal parameters under experimental conditions in three independent replicates, the average value of the biosynthesized theaflavin concentration reached 0.75±0.017mgml^–1 and matched the value predicted by the modelRevisions requested 03 November 2004; Revisions received 7 December 2004     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

Stereoselectivity of the reduced folate carrier in Caco-2 cells

Stereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco-2 cells using methotrexate (l-amethopterin or l-MTX) and its antipode (d-amethopterin or d-MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a K(m) value of approximately 0.6 microM. The Eadie-Hofstee plot of the RFC1-mediated FA uptake revealed a single component for FA uptake into Caco-2 cells, demonstrating that only RFC1 is involved in FA uptake. l-MTX inhibited FA uptake in a competitive manner with a K(i) value of approximately 2 microM, similar to the K(m) value of l-MTX. d-MTX also competitively inhibited FA uptake with a K(i) value being approximately 120 microM, indicating that the affinity of d-MTX is ca. 60-fold less than that of l-MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans.     

Emission mechanism of floral scent in Petunia axillaris

The mechanism of floral scent emission was studied in Petunia axillaris, a plant with a diurnal rhythm of scent output. The emission rate of each volatile compound oscillated in synchrony with its endogenous concentration, so that the intensity of the floral scent appeared to be determined by the endogenous concentrations. The composition of major volatiles in the flower tissue and the flower headspace showed characteristic differences. A negative correlation was found between the boiling points of the volatile compounds and the ratio of their emitted and endogenous concentrations, indicating that the composition of the floral scent depends directly on the endogenous composition of the volatile compounds. We conclude that in P. axillaris, the physiological regulation of floral scent emission operates not in the vaporization process but in the control of the endogenous concentrations of volatiles through biosynthesis and metabolic conversion.     

Control of body size by SMA-5, a homolog of MAP kinase BMK1/ERK5, in C. elegans

We have analyzed the sma-5(n678) mutant in C. elegans to elucidate mechanisms controlling body size. The sma-5 mutant is very small, grows slowly and its intestinal granules look abnormal. We found a 15 kb deletion in the mutant that includes a 226 bp deletion of the 3' end of the W06B3.2-coding sequence. Based on this result, rescue experiments, RNAi experiments and a newly isolated deletion mutant of W06B3.2, we conclude that W06B3.2 is the sma-5 gene. The sma-5 mutant has much smaller intestine, body wall muscles and hypodermis than those of the wild type. However, the number of intestinal cells or body wall muscle cells is not changed, indicating that the sma-5 mutant has much smaller cells. In relation to the smaller cell size, the amount of total protein is drastically decreased; however, the DNA content of the intestinal nuclei is unchanged in the sma-5 mutant. The sma-5 gene is expressed in intestine, excretory cell and hypodermis, and encodes homologs of a mammalian MAP kinase BMK1/ERK5/MAPK7, which was reported to control cell cycle and cell proliferation. Expression of the sma-5 gene in hypodermis is important for body size control, and it can function both organ-autonomously and non-autonomously. We propose that the sma-5 gene functions in a MAP kinase pathway to regulate body size mainly through control of cell growth.     

Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli

FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1, is a dimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to homology modeling, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain. A long alpha3 helix spans these domains. An N-domain with the entire alpha3 helix (N-domain+) and a C-domain with the entire alpha3 helix (C-domain+) were overproduced in Escherichia coli in a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein. C-domain+ was shown to be a monomer and enzymatically active. Its optimum temperature for activity (10 degrees C) was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in substrate-binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5 degrees C and 46.6 degrees C for the intact protein, and single heat absorption peaks at 44.7 degrees C for N-domain+ and 35.6 degrees C for C-domain+. These results indicate that the thermal unfolding transitions of the intact protein at lower and higher temperatures represent those of C- and N-domains, respectively. Because the unfolding temperature of C-domain+ is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibility around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22.