The induction of α-amylase activity by sucrose starvation in suspension-cultured rice cells is regulated by polyamines

When suspension-cultured rice ( Oryza sativa L. cv. Tainan 5) cells were deprived of sucrose, α-amylase (EC 3.2.1.1) activity in the cells and the culture medium increased markedly. The increase in activity of α-amylase caused by sucrose starvation in the cells and the medium was strongly reduced in the presence of exogenously added spermine. Putrescine and spermidine also inhibited, though only slightly, the increase in α-amylase activity caused by sucrose starvation. Preincubation of the enzyme extract or enzyme in the medium with polyamines had no effect on α-amylase activity. Sucrose starvation resulted in lower polyamine levels in rice suspension cells. D-Arginine and α-methylomithine, inhibitors of polyamine biosynthesis, caused reduced levels of polyamines and increased activity of α-amylase in rice suspension cells cultured in the presence of sucrose. Our results indicate that the induction of α-amylase activity by sucrose starvation in rice suspension cells is mediated, at least partly, through the internal level of polyamines.     

Mode of high temperature injury to wheat. II. Comparisons of wheat and rice with and without inflorescences

High temperature injury to wheat ( Triticum aestivum L.) during grain development is manifested as acceleration of senescence. Experiments were conducted to elucidate the mode of senescence and site of high temperature responses. Wheat (cv. Chris) and rice ( Oryza sativa L. cv. Newbonnet), which have C₃ photosynthesis but different temperature responses, were grown with and without inflorescences under three temperature regimes after anthesis. Plant growth and constituents associated with senescence were measured weekly until physiological maturity. Increasing temperatures from 25°C/15°C to 35°C/25°C day/night after anthesis decreased growth, leaf viability, chlorophyll and protein concentrations, and RuBP carboxylase activity and increased protease and RNase activities in wheat. Inflorescence removal increased vegetative weights and slowed most senescence processes more in wheat than in rice, but did not alter the course of high temperature responses. Results are interpreted as indicating that diversion of nutrients from roots by inflorescence sinks at normal temperatures and by increased respiration at high temperatures caused similar responses. Source and sink activities appeared to be regulated jointly, probably by cytokinins from roots, during senescence at normal and elevated temperatures.     

Effects of spermidine on the synthesis of α-amylase in cotyledons of mung bean seedlings

When cotyledons of mung bean [ Vigna radiata (L.) Wilczek] were treated with spermidine (3 m M ) during the first 6 h of imbibition, the development of α-amylase activity in cotyledons during the following 3 days was severely inhibited (75%) This inhibition was due to a slower accumulation of α-amylase protein, which in turn resulted from an inhibition of α-amylase synthesis. The rise in the level of α-amylase mRNA in cotyledons was also inhibited by spermidine treatment. However, the degree of inhibition of mRNA accumulation (40%) was not so marked as that of the activity of α-amylase synthesis (80%). These results are discussed in relation to the mode of action of spermidine on α-amylase expression.     

Localisation of β-oxidation enzymes in peroxisomes of rice coleoptiles

Enzymes of the β-oxidation pathway in rice ( Oryza sativa L., cv. Arborio) coleoptiles were investigated. The coleoptiles contain acyl-CoA oxidase (EC 1.3.99.3), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), enoyl-CoA hydratase (EC 4.2.1.17) and thiolase (EC 2.3.1.9). Analysis of coleoptile homogenates by sucrose density fractionation showed a preferential distribution of these enzymes in the unspecialized peroxisomes. The enzymatic activity found in the mitochondrial fraction was due to peroxisomal contamination since electron micrographs show the peroxisomes to be intact and pure whereas the mitochondrial fraction was contaminated by other organelles. It appears that the β-oxidation pathway is localized in the unspecialized peroxisomes of rice coleoptiles, extending the number of plant species in which such a localization has been observed.     

Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis

Abstract The gene coding for the thermostable α-amylase Bacillus licheniformis has been isolated from a direct shotgun in Escherichia coli using the bacteriophage lambda as a vector. The fragment containing the α-amylase gene has been sub-cloned in pBR322 and its restriction map determined. The α-amylase produced by the E. coli clones retained the thermostability of the B. licheniformis enzyme. Expression and properties of the gene product in E. coli and Bacillus subtilis have been examined.     

Purification of a β-galactosidase from rice shoots and its involvement in hydrolysis of the natural substrate in cell walls

Several glycosidase and glycanase activities have been detected in homogenates of rice ( Oryza sativa L. cv. Nipponbare) shoots after successive extraction with K-phosphate (pH 7. 0) and buffer containing 3 M LiCl. The major β-D-galactosidase (EC 3. 2. 1. 23) present in the buffer-soluble protein fraction was purified to electrophoretic homogeneity by a combination of chromatographic techniques including DEAE-Sepharose CL-6B, Sephacryl S-200HR and p -aminophcnyl-β-D-thiogalactopyranoside–Sepharose 4B. Analysis by denaturing gel electrophoresis revealed a single polypeptide chain with an apparent molecular mass of 42 kDa. Similar to the value of 40 kDa estimated for the native protein by gel-permeation. The isoelectric point was pH 6. 0. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyra-noside were 0. 63 m M and 0. 32 mmol (mg protein)⁻¹ h⁻¹, respectively. Maximum activity in McIlvaine buffer occurred at pH 3. 4, and the activity was inhibited by Ag²⁺, Cu²⁺. Hg²⁺, p -chloromercuribenzoate (PCMB) and D-galactono-l,4-lactone. The enzyme hydrolyzed larchwood arabinogalactan in an exo-fashion, and acted weakly on arabinosyl and galactosyl residue-rich polymer of pectic polysaccharides and cell walls from rice shoots.     

Cloning and expression of the thermostable α-amylase gene from Clostridium thermosulfurogenes (DSM 3896) in Escherichia coli

Abstract The gene coding for a thermostable α-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulforogenes carrying the α-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

Protein Kinase A Phosphorylation of the Proteasome: A Contribution to the α-Secretase Pathway in Human Cells

Abstract: The β-amyloid precursor protein undergoes a physiological cleavage by α-secretase that leads to the release of a secreted C-terminally truncated fragment called APPα and likely concomitantly reduces the formation of the amyloidogenic Aβ peptide. Here we demonstrate that APPα secretion is increased by the protein kinase A (PKA) effectors 8-bromo cyclic AMP and forskolin in human embryonic kidney cells (HK293), and that this can be prevented by a proteasome inhibitor. Furthermore, we establish that PKA effectors but not protein kinase C agonists increase the chymotrypsin-like activity and phosphorylation state of the proteasome in vitro and in vivo in HK293 cells. Altogether, this report demonstrates that the α-secretase pathway is under the control of PKA in human cells and that the proteasome likely contributes, either directly or through yet unknown intermediates, to the PKA-stimulated APPα secretion in human cells.     

Enhanced UV-B radiation has little effect on growth, δ13C values and pigments of pot-grown rice (Oryza sativa) in the field

Predicted increase in ultraviolet-B (UV-B: 280–320 mn) radiation may have adverse impacts on growth and yield of rice ( Oryza sativa L.), as has been found in studies hitherto. However, most of the studies were conducted in growth chambers or greenhouses where the plants are generally more sensitive to UV-B than in the field, presumably because of the distorted balance between UV-B and ultraviolet-A as well as PAR. This study was conducted to address the effects of enhanced UV-B on growth and yield of rice under a realistic spectral balance in the field. Three cultivars, "Koshihikari",'IR 45'and'IR 74'were pot-grown and irradiated with enhanced UV-B for most of the growing season in the field at Tsukuba, Japan (36°01'N, 140°07'E). The UV-B enhancement simulated ca 38% depletion of stratospheric ozone at Tsukuba. The results showed no UV-B effects on plant height, numbers of tillers and panicles, dry weight of the plant parts or the grain yield for any of the 3 cultivars. Natural abundance of ¹³C in the flag leaves was not altered by the UV-B enhancement either. While UV-absorbing compounds showed no response to the UV-B enhancement, chlorophyll contents decreased with enhanced UV-B. However, the decrease of chlorophyll was limited to an early growth stage with no effect later. We thus found no extraordinary impact of the nearly doubled UV-B radiation on rice in the field, and it would appear that a reliable prediction of the effects of UV-B will require experiments carried out over a number of years under various climatic and solar UV-B regimes.     

Noradrenergic Inhibition and α2-Adrenergic Stimulation of Melatonin Secretion in the Pigeon

Adrenergic regulatory mechanisms of melatonin synthesis and secretion were studied in the pigeon in vivo. Late-afternoon intraperitoneal injection of noradrenaline (NA; 1 mg/kg) resulted in a significant decrease in plasma melatonin levels in 3 h. The same effect was seen after phenylephrine treatment (1 mg/kg i.p.), indicating that an alpha 1-adrenergic mechanism may mediate the inhibition. Propranolol treatment had no effect on plasma melatonin levels, supporting this concept. Detomidine (1 mg/kg i.p.), an alpha 2-adrenergic agonist, increased melatonin levels. This stimulatory effect was blocked by yohimbine, an alpha 2-adrenergic antagonist. However, yohimbine alone had no effect on the plasma melatonin levels, suggesting that alpha 2-adrenergic transmission is not primarily responsible for the nocturnal stimulation of melatonin synthesis and secretion in the pigeon.     

Presence of β-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination

Evolution of HCN from both rice ( Oryza sativa ) and cocklebur ( Xanthium pennsylvanicum ) seeds increased during a pre-germination period and preceded the evolution of (C₂H₄). These two species were adopted as the representatives of starchy and fatty seeds, respectively. Ethylene promotes seed germination of many species. However, HCN evolution declined abruptly when the radicles emerged and before the peak in C₂H₄ evolution. More-over, both rice and soybean ( Glycine max ) seeds showed some activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9) even in the unimbibed dry state. The activities of CAS in the lower seed of cocklebur and in soybean seeds increased rapidly after emergence of the radicle. However, the CAS of rice seeds, with high activity in the dry state, exhibited a bimodal change, gradually decreasing until radicle emergence had occurred, but then increaing. It is thus likly that HCN evolution during initial imbibition may be derived from cyanogenic reserves and controlled by both pre-existing and subsequently-developing CAS. The exogenous application of C₂H₄ stimulated the activities of CAS in both rice and upper cocklebur seeds and reduced their cyanogen contents. Therefore, the decline of HCN evolution after germination seems to be due to the increased activities of CAS by endogenously produced C₂H₄.     

β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

Calcium Regulation of Agonist Binding to α7-Type Nicotinic Acetylcholine Receptors in Adult and Fetal Rat Hippocampus

Abstract: Quantitative autoradiography was used to compare the binding properties of α7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of ¹²⁵I-α-bungarotoxin (¹²⁵I-α-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of ¹²⁵I-α-BTX binding, as well as α-cobratoxin and nicotine potency to displace ¹²⁵I-α-BTX, did not change with age. Addition of Ca²⁺ to the assay buffer did not alter ¹²⁵I-α-BTX binding, or α-cobratoxin inhibition of ¹²⁵I-α-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca²⁺ on agonist affinity was dose-dependent, with an EC₅₀ value of 0.25–0.5 m M . Ca²⁺ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal ¹²⁵I-α-BTX binding sites are largely similar across age. Ca²⁺ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.     

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin α-amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems

Aims:  To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems.
Methods and Results:  Our program to screen for new α-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus , for which we propose the name S. coelicoflavus var. nankaiensis . Four chemically distinct α-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas α-amylase inhibitors. Acarviostatin III03 is the most potent α-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood.
Conclusions:  Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective α-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes.
Significance and Impact of the Study:  Acarviostatin III03 is the most potent α-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between α-amylase and various inhibitors and will offer more choices in diabetes treatments.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

Effects of aluminium on growth and kinetics of K+(86Rb) uptake in two cultivars of wheat (Triticum aestivum) with different sensitivity to aluminium

Two cultivars of wheat (Triticum aestivum L. cvs Kadett and WW 20299) were grown for 9 days with 20% relative increase in nutrient supply per day at pH 4.1. Aluminium at 50 μ M retarded the growth of roots more than that of shoots in both cultivars, thus decreasing the root/shoot ratio. The inhibition was largest in WW 20299. With long term Al treatment (9 days), K_m for K⁺(⁸⁶Rb) influx increased five times in both cultivars and V_max decreased in WW 20299. Efflux of K⁺(⁸⁶Rb) was little affected. When the roots were treated with aluminium for two days, only relative growth rate of roots was retarded, while growth of shoots was unaffected and influx of K⁺(⁸⁶Rb) adjusted to the actual K⁺ demand of the plants. It is concluded that the effects of aluminium on K⁺ uptake in these wheat cultivars are not primary factors contributing to aluminium sensitivity. However, in soil with Al the demand for a comparatively high concentration of K⁺ to maintain an adequate K⁺ uptake rate, in combination with a slow growth rate of the roots, may secondarily lead to K⁺ deficiency in the plants.