Expression of fungal thermotolerant endo-1,4-β-glucanase in transgenic barley seeds during germination

The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4--glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI -amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 °C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble -glucan content. A decrease in the soluble -glucan content in the wort improves the filtration rate of beer.     

Production,properties, and applications of endo-β-mannanases

The present review provides up-to-date information on the occurrence and methodologies used for producing and purifying endo-β-mannanases and a comprehensive comparison of their biochemical properties. The amalgamation of biochemical, molecular and structural biology approaches which have been used for understanding endo-β-mannanase families, catalytic mechanism, substrate binding, non-catalytic modules, trans-glycosylation, and multi-functional enzyme complexes has been given critical attention. A separate section entailing the state-of-the-art about thermostable endo-β-mannanases, which has emerged as an exciting field of both basic and applied research, is also deliberated. The remarkable progress made by endo-β-mannanases in various industrial sectors like food, feed, detergents, biofuel and oil drilling is also emphasized.     

A rapid response of β-amylase to nitric oxide but not gibberellin in wheat seeds during the early stage of germination

The effects of nitric oxide (NO) and gibberellic acid (GA₃) on the responses of amylases in wheat (Triticum aestivum L.) seeds (caryopses) were investigated during the first 12 h of germination. GA₃ had no effects on the activities of -amylase (EC 3.2.1.1) or -amylase (EC 3.2.1.2), either in intact seeds or embryoless halves within 12 h. In contrast, addition of sodium nitroprusside (SNP), an NO donor, was able to induce a rapid increase in -amylase activity without affecting -amylase. Furthermore, the rapid response of -amylase to SNP in wheat seeds could be attributed to NO and was approximately dose-dependent. Some other aspects of SNP induction of amylase isozymes were also characterized. Further investigations showed that SNP might play an interesting role in the dissociation of free -amylase from small homopolymers or heteropolymers. Furthermore, SNP also directly induced the release of bound -amylase from glutenin and its crude enzyme preparation. However, the slight increase in protease also induced by SNP might not be responsible for this action. Interestingly, based on the fact that the rapid response of -amylase to NO also existed in seeds of other species, such as barley, soybean, rice and watermelon, it might be a universal event in early seed germination.     

β-Tubulin accumulation and DNA synthesis are sequentially resumed in embryo organs of cucumber (Cucumis sativus L.) seeds during germination

Summary Resumption of DNA synthetic activities and -tubulin accumulation was studied in embryo organs of germinating cucumber (Cucumis sativus L.) seeds. Flow-cytometric analysis indicated the existence of 2C, 4C, and 8C nuclei in the radicle of mature embryos, whereas in cotyledons most of the cells contained nuclei with 2C DNA content. Upon imbibition of water, nuclear DNA replication was initiated in the radicle within 15 h, subsequently spreading towards the cotyledons. Bromodeoxyuridine incorporation preceded detectable changes in the relative amounts of DNA, implying the occurrence of putative DNA repair. Organellar DNA synthesis occurred independently of the nuclear DNA synthetic cycle. Western blotting and immunohistochemical localization demonstrated that the constitutive level of -tubulin originated from preserved -tubulin granules. During imbibition, disappearance of fluorescent tubulin granules, accumulation of -tubulin, and formation of microtubular cytoskeleton were found in the radicle, but not in the cotyledon areas. Mitosis only occurred after radicle protrusion at 21 h of imbibition. It is concluded that the differences in the initiation and progress of these cellular and molecular events are associated with the discrete behaviors of the radicle and the cotyledons upon imbibition. The formation of cortical microtubular cytoskeleton and the accumulation of tubulins are important features in preparation of radicle protrusion, whereas DNA synthesis may contribute to postgerminative growth.     

Hydrogen sulfide stimulates β-amylase activity during early stages of wheat grain germination

We recently reported that H₂S could significantly promote the germination of wheat grains subjected to aluminum (Al³⁺) stress.¹ In these experiments seeds were pretreated with the H₂S donor NaHS for 12 h prior to Al³⁺ stress. During this pre-incubation period we observed that H₂S increased the activity of grain amylase in the absence of Al³⁺. Using embryoless half grains of wheat we now show that H₂S preferentially affects the activity of endosperm β-amylase and that α-amylase synthesis and activity is unaffected by this treatment.Key words: α-amylase, β-amylase, hydrogen sulfide, reactive sulfur species, seed germination, wheat (triticum)Cereal grains contain many acid hydrolases, some synthesized de novo by the scutellum and aleurone layer and others are found preformed in the starchy endosperm. The amylases are the best known of these types of enzymes. α-Amylases are synthesized and secreted by the scutellum and aleurone layer, and in the case of aleurone isoforms their synthesis is regulated by GAs and ABA.^2,3 Whereas GAs stimulate the synthesis of α-amylases and many other secreted hydrolases, ABA inhibits these processes. β-Amylases, on the other hand, are preformed enzymes whose synthesis is not affected by ABA and GAs. Two forms of β-amylase are found in wheat grains, one is a soluble form present in ungerminated grains which can form high-molecular-weight homopolymers or heteropolymers; the other is bound in an inactive form via S-S linkages to proteins at the periphery of starch grains.^4,5 The activation of β-amylases is thought to result at least in part from their release from endosperm proteins by the action of GA-induced proteases secreted from the aleurone layer.⁶To examine in more detail the effect of H₂S on wheat grain amylases we used de-embryontated wheat grains where only the aleurone layer and starchy endosperm were the possible sources of amylase activity. Embryoless half grains of wheat (Triticum aestivum L., c.v Yangmai 158) were incubated in water, the H₂S donor NaHS, GA, or combinations of these treatments for up to 12 h. After incubation soluble proteins were isolated from grains by homogenizing in buffer and amylase activity was determined colorimetrically using starch-I₂KI. Figure 1A shows that during the first 8 h of incubation there was no increase in amylase activity from half grains incubated in H₂O or GA but there was a small but significant increase in activity after 10 h and 12 h of incubation in these two treatments. By contrast, incubation in NaHS brought about a three-fold increase in amylase activity by six h and this increased to about five-fold above the initial activity by 12 h of incubation.Open in a separate windowFigure 1Amylolytic activity from wheat half grains measured colorimetrically (A), by diffusion into agar-starch (B) and following native gel electrophoresis (C and D). (A) Half grains were incubated in water (CK), 0.5 mM NaHS, 10 µM GA3 and 0.5 mM NaHS plus 10 µM GA3 for up to 12 h. Amylase activity was measured colorimetrically on grain homogenates by the starch I-KI method. (B) Half grains pre-incubated as (A) then transferred to agar containing 0.2% starch and 2 mM EDT A for a further 12 h. Amylolytic activity was determined by the diameter of the starch-free halo after flooding the agar-starch plate with I-KI solution. Seeds treated with CK, NaHS, GA₃ and NaHS plus GA₃ were lined from left to right in each plate, respectively. (C and D) Wheat grains incubated for 12 h in H₂O, NaHS, GA₃ and Hb (0.1 g/L) and combinations of these treatments, were homogenized and aliquots of extracts were electrophoresed by non-denaturing PAGE. After electrophoesis gels were soaked in starch, washed and stained with I-KI. Amylolytic activity is shown by cleared areas in the gel. For the gel in (C), homogentes were incubated with 25 mM EDT A to inactivate α-amylase and for (D), they were heated at 70°C to inactivate β-amylase.We confirmed these results by incubating wheat half gains on 0.2% agar containing 0.2% soluble starch and 2 mM EDTA (Fig. 1B). In this experiment, half grains were first incubated in H₂O, GA or NAHS for up to 12 h as for the experiment shown in Figure 1A and half grains were transferred to agar to estimate amylase activity. Because α-amylases are Ca²⁺-containing metalloenzymes whose activities are dependent on Ca²⁺ binding we incorporated EDTA into the agar to favor the appearance of β-amylase activity. The data in Figure 1B confirm what we observed when we measured amylase activity colorimetrically, namely that starch degrading activity was high in half grains exposed to NaHS but low in those incubated in H₂O or GA. From this experiment we also concluded that the starch degrading activity was likely a result of the activity of β-amylase as α-amylase activity would have been reduced or eliminated by the presence of EDTA.We established that the amylolytic activity produced by wheat grains in response to the NaHS donor was largely β-amylase by selectively inhibiting amylase activities following native gel electrophoresis. α-Amylases are heat stable but are sensitive to metal chelators, whereas β-amylases are denatured by heating but are not inhibited by chelators such as EDTA.⁴ Figure 1C and D show the activities of amylase isoforms measured by incubating non-denaturing polyacrylamide gels in 1% soluble starch followed by staining in I-KI. Figure 1C shows amylolytic activity after incubation of the gel in starch containing 25 mM EDTA. Two distinct sets of bands are seen together with minor bands of activity. Whereas group I amylases do not change significantly following treatment incubation in the presence of NaHS or GA, group II amylases show high activity in the presence of NaHS, but show no activity with GA (Fig. 1C). When the enzyme preparations were heated to 70°C for 15 min before electrophoresis almost all activity was lost showing that the activity seen in the absence of heating but in the presence of EDTA was likely that of β-amylase. We also used hemoglobin (Hb) a nonspecific H₂S scavenger to show that the effects of NaHS were indeed via the production of H₂S. Figure 1C shows that Hb almost completely abolished the inductive effect of NaHS on β-amylase activity.Although at present we have no direct evidence that H₂S acts as an endogenous regulator of endosperm function in cereal grains, it is tempting to speculate that this is indeed the case. Our previous work showed an increase in the synthesis of H₂S by wheat grains that is detected as early as 12 h following the start of imbibition in H₂O.¹ Although we have no information on the route of H₂S synthesis in wheat, there is abundant evidence that plants synthesize H₂S as its emission has been observed in many species.^7–13 In plants, H₂S is thought to be released from cysteine via a reversible O-acetylserine (thiol) lyase (OASTL) reaction, and recently several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially characterized from Arabidopsis thaliana, confirming H₂S release by the action of desulfhydrases in various cellular compartments.^14–17 The induction of L-cysteine desulfhydrase upon pathogen attack,¹⁸ freezing tolerance by H₂S fumigation,¹⁹ emission of H₂S from plants exposed to SO₂ injury,^10,20 and abiotic stresses tolerance in plants treated with H₂S donor,^1,21–24 all infer that H₂S is involved in these responses.It is also tempting to speculate that H₂S may work in cereal grains by influencing redox status.^15,25 It has been proposed that H₂S can bring about an increase in synthesis of glutathione from cysteine and an overall improvement of plant performance under stress. The release of β-amylase from starchy endosperm proteins or the dissociation of free β-amylase from small homopolymers or heteropolymers has been shown to be enhanced by reducing agents such as dithiothreitol and by S-H proteinases. We propose that one plausible action of H₂S in cereal endosperm is to enhance reactive sulfur species that can lead to reduction of S-S bonds between β-amylase and its binding partners in the endosperm. Release of activated β-amylase would then be free to participate in starch degradation providing sugar units for seedling growth and development prior to the induction of α-amylases by GAs.     

Can rodents enhance germination rates in rainforest seeds?

The decline of large coevolved frugivorous species within fragmented habitats can have an effect on ecological processes, for example, seed dispersal and germination. It is therefore necessary for more resilient species to ensure essential processes are maintained within the system. This study investigates the influence of two rodent species, Melomys cervinipes (Fawn‐footed Melomys) and Rattus fuscipes (Bush Rat), on the germination process of rainforest fruits. Both species are endemic to north Queensland rainforest and commonly found in fragmented habitats in high densities. We found in 85% of fruit species tested, rodent feeding increased seed germination rate by a factor of 3.5. Our results suggest that rodents can play a significant role in enhancing germination rates of fruits in the tropical rainforest of far north Queensland.     

Isolation and partial characterisation of α-amylase components evolved during early wheat germination

The development of α-amylase (EC 3.2.1.1) activity in wheat was followed during 4 days of germination. The enzyme was purified and separated by gel chromotography into two distinct entities (α-amylase I and α-amylase II), with different molecular weights and isoelectric points. α-Amylase I contained a much higher content of sugars than α-amylase II, which decreased as the germination proceeded. The time sequence analysis of the starch degradation pattern showed that on the 4th day of germination, 15% of the total activity was present in α-amylase I and the rest in a-amylase II. Similarly, differences in the relative rates of synthesis of their isoenzymes were observed. α-Amylase I was resolved on the 4th day of germination, only into 3 isoenzymes, whereas α-amylase II could separate into 4 isoenzymes. The enzyme activity was however maximal in the most electropositive isoenzyme in both the components.     

Re-interpreting the role of endo-��-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall

Background

Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose–cellulose network with a similar function to xyloglucans. Endo-β-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-β-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-β-mannanases to be reinterpreted.

Aims

In this review, the role of endo-β-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported.

Future outlook

There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.Key words: Cell wall, endo-β-mannanase, endohydrolase, mannan, endotransglycosylase     

Mannans in tomato fruit are not depolymerized during ripening despite the presence of endo-β-mannanase

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-β-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-β-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-β-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-β-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe.     

β-Pinene inhibited germination and early growth involves membrane peroxidation

β-Pinene, an oxygenated monoterpene, is abundantly found in the environment and widely occurring in plants as a constituent of essential oils. We investigated the phytotoxicity of β-pinene against two grassy (Phalaris minor, Echinochloa crus-galli) and one broad-leaved (Cassia occidentalis) weeds in terms of germination and root and shoot growth. β-Pinene (0.02–0.80 mg/ml) inhibited the germination, root length, and shoot length of test weeds in a dose–response manner. The inhibitory effect of β-pinene was greater in grassy weeds and on root growth than on shoot growth. β-Pinene (0.04–0.80 mg/ml) reduced the root length in P. minor, E. crus-galli, and C. occidentalis over that in the control by 58–60, 44–92, and 26–85 %, respectively. In contrast, shoot length was reduced over the control by 45–97 % in P. minor, 48–78 % in E. crus-galli, and 11–75 % in C. occidentalis at similar concentrations. Further, we examined the impact of β-pinene on membrane integrity in P. minor as one of the possible mechanisms of action. Membrane integrity was evaluated in terms of lipid peroxidation, conjugated diene content, electrolyte leakage, and the activity of lipoxygenases (LOX). β-Pinene (≥0.04 mg/ml) enhanced electrolyte leakage by 23–80 %, malondialdehyde content by 15–67 %, hydrogen peroxide content by 9–39 %, and lipoxygenases activity by 38–383 % over that in the control. It indicated membrane peroxidation and loss of membrane integrity that could be the primary target of β-pinene. Even the enhanced (9–62 %) activity of protecting enzymes, peroxidases (POX), was not able to protect the membranes from β-pinene (0.04-0.20 mg/ml)-induced toxicity. In conclusion, our results show that β-pinene inhibits root growth of the tested weed species through disruption of membrane integrity as indicated by enhanced peroxidation, electrolyte leakage, and LOX activity despite the upregulation of POX activity.     

Post-translational processing of β-d-xylanases and changes in extractability of arabinoxylans during wheat germination

Endo-1,4-β-d-xylanase (EC 3.2.1.8, β-d-xylanase) activity, and arabinoxylan (AX) level and extractability were monitored for the first time simultaneously in wheat kernels (Triticum aestivum cv. Glasgow) up to 24 days post-imbibition (DPI), both in the absence and presence of added gibberellic acid (GA). Roughly three different stages (early, intermediate and late) can be discriminated. Addition of GA resulted in a faster increase of water extractable arabinoxylan (WEAX) level in the early stage (up to 3–4 DPI). This increase was not accompanied by the discernible presence of homologues of the barley X-I β-d-xylanase as established by immunodetection. This suggests that other, yet unidentified β-d-xylanases operate in this early time window. The intermediate stage (up to 13 DPI) was characterized by the presence of unprocessed 67 kDa X-I like β-d-xylanase, which was much more abundant in the presence of GA. The occurrence of higher levels of the unprocessed enzyme was related with higher β-d-xylanase activities and a further increase in WEAX level, pointing to in vivo activity of the unprocessed 67 kDa β-d-xylanase. During the late stage (up to 24 DPI) gradual processing of the 67 kDa β-d-xylanase occurred and was associated with a drastic increase in β-d-xylanase activity. Up to 120-fold higher activity was recorded at 24 DPI, with approx. 85% thereof originating from the kernel remnants. The WEAX level decreased during the late stage, suggesting that the β-d-xylanase is processed into more active forms to achieve extensive AX breakdown.     

Characterization of tomato endo-β-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection

The tomato (Lycopersicon esculentum Mill.) endo--1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.This revised version was published online in August 2004 with corrections to Fig. 1 and Fig. 5.     

Immunolocalization of estrogen receptor α in Neomysis japonica oocytes and follicle cells during ovarian development

Estrogen induces oocytes development and vitellogenesis in crustacean by interacting with estrogen receptor (ER) subtypes. In the present study, we detect for the first time the ERα in oocytes and follicle cells and hepatopancreas cells of mysis by immunohistochemistry using a specific ERα antibody. ERα was mainly localized in the nuclei of oocytes and follicle cells, while mainly detected in nuclei of oogonia (OG), previtellogenic oocyte (PR) and endogenous vitellogenic oocyte (EN) at previtellogenic and early vitellogenic stage (I-early III). Follicle cells in all stages of ovary (all vitellogenic stages) showed strong ERα positive reaction, and they were able to gradually move to oocytes during the development of oocytes. In addition, ERα was also localized in the nuclei and cytoplasm of four hepatopancreas cells (including E-, R-, F- and B-cell) in all ovary stages. These findings suggest, for the first time to our knowledge, that there could be a close link between oogenesis, follicle cells, hepatopancreas cells and endocrine regulation, and estrogens might be involved in the regulation of oocytes at early ovarian stage in mysis.     

Effect of abscisic acid on galactomannan degradation and endo-β-mannanase activity in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the effect of abscisic acid (ABA) on galactomannan degradation during and after germination. Seeds were imbibed in water or in 10⁻⁴ M ABA, and used to evaluate the effect of exogenous and endogenous ABA. Tissue printing was used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. The presence of exogenous ABA provoked a delay in the cellular disassembly of the endosperm and disappearance of endo-β-mannanase in the tissue. This led to a delay in galactomannan degradation. The testa (seed coat) of S. virgata contains endogenous ABA, which decreases ca. fourfold during storage mobilisation after germination, permitting the galactomannan degradation in the endosperm. Furthermore, endo-β-mannanase was immunolocalised in the testa, which has a living cell layer. The ABA appears to modulate storage mobilisation in the legume seed of S. virgata, and a cause–effect relationship between ABA (probably through testa) and activities of hydrolases is proposed.     

Endo-β-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo--mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo--mannanase in the endosperm after germination is induced by GAs, but the expression of endo--mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo--mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo--mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.Abbreviations ABA cis/trans-abscisic acid - GA(s) gibberellin(s) - IEF isoelectric focussing - pI(s) isoelectric point(s) We thank Dr. Bruce Downie for the seemingly endless but inspiring discussions.     

Amyloid-β: the seeds of darkness

Whilst the amyloid-β (Aβ) hypothesis/centric theory continues to evolve, genetic, biochemical and pathological evidence still suggests that Aβ is central to the etiology of Alzheimer's disease (AD). In particular, Aβ-oligomers/soluble Aβ, may be an earlier determinant of Alzheimer's disease and better correlative of cognitive impairment. Whilst there are a number of Aβ-oligomeric species in existence (making therapeutic and diagnostic biomarker choice cumbersome), their existence is in equilibrium with Aβ-fibrils, the main constituent of cored plaques. Although Alzheimer's disease remains incurable, improvements to Aβ immunotherapies and strategies to target Aβ continue to evolve, with the reliance upon Aβ imaging to shed light on the outcome of therapeutics proving very useful.     

β-galactosidase activity in the germinating seeds of Vigna sinensis

The β-galactosidase activity in cotyledons of Vigna sinensis increases during seed germination and is inhibited by cycloheximide. The increasing activity may be due to the de novo synthesis of enzyme protein. The enzyme has been partially purified by gel filtration and characterized with respect to some biochemical parameters. The optimum pH and optimum temperature are 4.5 and 55°, respectively and the enzymes follows typical Michaelis kinetics with K_m and V_max of 4.5 x 10^?4 M and 2.0 x 10^?5 mol/hr respectively. K_i for galactose and lactose are 4.5 and 220 mM, respectively. The energy of activation of the enzyme for p-nitrophenyl β-D-galactoside is 9.83 kcal/mol. The apparent relative MW of the enzyme as determined by gel filtration was 56000.