Variation in ruminal in situ degradation of crude protein and starch from maize grains compared to in vitro gas production kinetics and physical and chemical characteristics

The objectives of this study were (1) to evaluate in situ ruminal dry matter (DM), crude protein (CP) and starch degradation characteristics and in vitro gas production (GP) kinetics using a set of 20 different maize grain genotypes and (2) to predict the effective degradation (ED) of CP and starch from chemical and physical characteristics alone or in combination with in vitro GP measurements. Maize grains were characterised by different chemical and physical characteristics. Ruminal in situ degradation was measured in three lactating Jersey cows. Ground grains (sieve size: 2 mm) were incubated in bags for 1, 2, 4, 8, 16, 24, 48 and 72 h. Bag residues were analysed for CP and starch content. Degradation kinetics was determined and the ED of DM, CP and starch calculated using a ruminal passage rate of 5%/h and 8%/h. The GP of the grains (sieve size: 1 mm) was recorded after 2, 4, 6, 8, 12, 24, 48 and 72 h incubation in buffered rumen fluid and fitted to an exponential equation to determine GP kinetics. Correlations and stepwise multiple linear regressions were evaluated for the prediction of ED calculated for a passage rate of 5%/h (ED5) for CP (EDCP5) and starch (EDST5). The in situ parameters and ED5 varied widely between genotypes with average values (±SD) of 64% ± 4.2, 62% ± 4.1 and 65% ± 5.2 for ED5 of DM, EDCP5 and EDST5 and were on average 10 percentage points lower for a passage rate of 8%/h. Degradation rates varied between 4.8%/h and 7.4%/h, 4.1%/h and 6.5%/h and 5.3%/h and 8.9%/h for DM, CP and starch, respectively. These rates were in the same range as GP rates (6.0–8.3%/h). The EDCP5 and EDST5 were related to CP concentration and could be evaluated in detail using CP fractions and specific amino acids. In vitro GP measurements and GP rates correlated well with EDCP5 and EDST5 and predicted EDCP5 and EDST5 in combination with the chemical characteristics of the samples. Equations can be used to obtain quick and cost effective information on ruminal degradation of CP and starch from maize grains.     

Stimulation of vitamin B12 formation in aerobically-grownRhodopseudomonas gelatinosa under microaerobic condition

Summary Photopigments and vitamin B12 formation ofRhodopseudomonas gelatinosa were enhanced by a stepwise change of the culture condition from aerobic (oxidation-reduction potential, ORP>+110 mV) to microaerobic condition (ORP=0 to –200 mV). During the microaerobic culture in the malateglutamate medium, -aminolevulinic acid synthetase (ALAS) increased 2 to 4 folds in 4 h with the increases in intracellular content of carotenoid, bacteriochlorophyll and vitamin B12. Effects of light illumination on vitamin B12 formation could not be observed. Further, the production of SCP enriched with vitamin B12 and photopigments from cassava starch was done by changing aerobic to microaerobic culture resulting that intracellular carotenoid, bacteriochlorophyll and vitamin B12 increased to 310, 960 and 38 g/g cell from 230, 0 and 25 g/g cell of aerobic culture, respectively.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Uptake, Distribution, and Metabolism of 1-Naphthaleneacetic Acid and Indole-3-Acetic Acid During Callus Initiation From Actinidia deliciosa Tissues

1-Naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) were required for in vitro callus formation at the basal edge of kiwifruit (Actinidia deliciosa [A. Chev] Liang and Ferguson, cv. Hayward) petioles. The uptake, metabolism, and concentration of NAA and indole-3-acetic acid (IAA) content were examined in the explants during the callus initiation period. After 1, 6, 12, 24, 48, and 96 h of culture in the presence of [H³]NAA, petioles were divided into apical, middle, and basal portions and analyzed. Except for a high IAA level measured at 12 h, IAA content decreased in tissues during a culture period of 96 h. NAA uptake was higher in petiolar edges than in the middle portion, and NAA was rapidly conjugated with sugars and aspartic acid inside the tissues. The amide conjugation was triggered in apical and basal portions from 12 h and in the middle part from 48 h, with α-naphthylacetylaspartic acid being the major metabolite. Free-NAA concentration in cultured petioles achieved an equilibrium with the exogenously applied NAA (0.27 μm) from 12 h, and it remained constant thereafter. The relationships between the role attributed to NAA and BA in the initiation and the maintenance of disorganized growth of callus in kiwifruit cultures are discussed. Received December 21, 1998; accepted July 20, 1999     

Cumulus cell‐enclosed oocytes acquire a capacity to synthesize GSH by FSH stimulation during in vitro maturation in pigs

We investigated (i) follicle stimulating hormone (FSH)‐modulated changes in the expression of glutathione (GSH) and its rate‐limiting enzyme, glutamate cysteine ligase (GCL), in porcine oocytes and cumulus cells, and (ii) the contribution of gap‐junctional communications (GJCs) in cumulus‐oocyte complexes (COCs) to intraoocyte GSH accumulation. In experiment (i), COCs were cultured for 48 h with (+FSH group) or without FSH (−FSH group). The GSH content of oocytes increased with cultivation time in the +FSH group, but decreased in the −FSH group. The GSH content of cumulus cells at 48 h was also higher in the +FSH group than that in the −FSH group. Expression of GCL subunit mRNAs in oocytes and cumulus cells was increased by FSH stimulation until 12 h, and then fell to the baseline level. On the other hand, the amount of GCL subunit proteins in oocytes and cumulus cells increased gradually throughout the period of culture with FSH. In experiment (ii), blocking of GJCs in COCs during 0–24 h of culture led to a decrease in the GSH content of oocytes at 24 h of culture, whereas the GSH content at 48 h of culture did not differ even after blocking of the GJCs during 24–48 h of culture. These findings indicate that FSH initiates GSH synthesis in cumulus cells and oocytes by modulating the expression of GCL, and that porcine oocytes are able to synthesize GSH without GJC‐mediated support from cumulus cells, at least in the later half of maturation culture. J. Cell. Physiol. 222: 294–301, 2010. © 2009 Wiley‐Liss, Inc.     

Microalgae--novel highly efficient starch producers

The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch‐rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale‐up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m² s¹), 40% of DW was synthesized at a mean light intensity 330 µmol/(m² s¹). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory‐scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide‐treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled‐up culture of starch‐enriched biomass, was carried out in an outdoor pilot‐scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW. Biotechnol. Bioeng. 2011; 108:766–776. © 2010 Wiley Periodicals, Inc.     

Changes in the carbohydrate content of the leaf and the apical bud of Sinapis during transition to flowering

Vegetative plants of Sinapis alba L. were induced to flower by a single long day of 20 h or by a single short day of 8 h starting at an unusual time of the 24-h cycle (displaced short day). The soluble sugar and starch contents of the just-expanded leaf and the apical bud were measured at various times after the start of each of these two photoinductive treatments. Associated with the induction of flowering there were temporary increases in the soluble sugar and starch contents of the leaf and of the bud. These increases were apparent 14 h after the start of the long day and 12 h after the start of the displaced short day. The starch content of the bud increased later. These results indicate that an increase of the soluble sugar content of the bud is required for its transition from the vegetative to the reproductive condition.     

Starch Measurement in Plant Tissue Using Enzymatic Hydrolysis

This work explored completeness of starch hydrolysis in situ in relation to degree of gelatinization, starch content of tissue, evailable enzyme activity, and time allowed for hydrolysis. Maximum hydrolysis of starch in lyophilized red oak (Quercus rubra L.) root tissue with purified Diazyme (amyloglucosidase) or Clarase (Takadiastase) required high enzyme activity (2.4 U Diazyme or 48 U Clarase per mg starch). Results suggested that at least 70 U Clarase or 5 U Diazyme should be used per mg starch in routine analyses. Neither prolonging gelatinization (more than 15 min) nor hydrolysis (more than 24 to 48 lh) offset inadequate starch hydrolysis caused by insufficient enzyme activity. Starch was completely hydrolyzed in situ after 48 h without gelatinization by 5 U of Diazyme per mg starch. Tissue weight (5 to 100 mg) had no effect on starch hydrolysis by sufficient enzyme. Methanol: chloroform: water (12:5:3 by volume) freed tissues of solubles before starch hydrolysis. No interference with the glucose oxidase analysis of hydrolysates was encountered. In addition, the pigment free methanol–water fractions (soluble sugars, amino acids, organic acids) and chloroform fractions (lipids and pigments) were available or further analysis. Results obtained with red oak were verified with issue from other species such as jack pine (Pinus banksiana lamb.) and white spruce (Picea glauca (Moench) Voss). The resulting technique simply and reliably measured less than 5% starch in 5 mg lyophilized tissue, with a minimum of sample manipulation.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

登革病毒对人树突状细胞感染性的研究

探讨登革病毒对人树突状细胞(DC)的感染性。人外周新鲜血常规分离单核细胞,经细胞因子GMCSF、IL4诱导培养成DC,通过形态学特征、细胞表型和淋巴细胞刺激能力鉴定。用登革病毒2型(DV2)感染DC,于作用后6h、24h、48h、72h、96h分别收集上清液和细胞,甲基纤维素微量空斑试验测定病毒滴度,间接免疫荧光法检测细胞上病毒抗原表达,透射电镜观察病毒在细胞内的定位。病毒感染后6h即可在培养上清中测出病毒,病毒滴度在48h达到高峰,以后逐渐下降。间接免疫荧光法证明感染的DC胞浆及胞膜上携带病毒抗原。透射电镜下在病毒感染48h后DC胞浆内可见大量病毒颗粒。树突状细胞是登革病毒感染的靶细胞,病毒可感染DC并产生大量病毒颗粒,可能在其发病机制中起重要作用。     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

Production of tropane alkaloids by transformed root cultures of Atropa belladonna in stirred bioreactors with a stainless steel net

A scale up of transformed root cultures of Atropa belladonna from a 300-ml flask to a 30-l tank was accomplished without any reduction in alkaloid productivity. Cutting treatment of seed cultures showed no distinct effect on root growth, morphology, and alkaloid content in conical flasks during 1 month of culture. Randomly cut roots thus grown were further cultivated in 3-l and 30-l modified stirred bioreactors for a scale-up culture. After 1 month of culture, 1490 mg of tropane alkaloids was produced by a 30-l culture of A. belladonna transformed roots. These roots contained the same level of atropine (5.4 mg/ g dw) as the roots of this plant grown in the field for 12 months and still contained a considerable amount of other alkaloids including 1.6 mg/g dw of 6-β-hydroxyhyoscyamine, 0.9 mg/g dw of scopolamine, and 2.0 mg/g dw of littorine. Received: 12 June 1998 / Revision received: 31 August 1998 / Accepted: 27 October 1998     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

Histological and scanning electron microscopic observations on plant regeneration in mungbean cotyledon (Vigna radiata (L.) Wilczek) cultured in vitro

Explanted cotyledons of mungbean Vigna radiata (L). Wilczek, variety Pag-asa-1, regenerated shoots directly from the basal adaxial side of the petiolar residue on MS medium supplemented with 8.9 M 6-benzyladenine. A simplified and rapid procedure for glycol methacrylate sectioning for histological observations was used to observe shoot initiation. At the time of culture, comparatively smaller and differentially stained epidermal cells were present on the basal adaxial region of the petiolar residue. A meristematic cell mass that developed at 48 h after culture appeared to be of epidermal and subepidermal cell origin. Scanning electron microscopy revealed shoot primordia and approximately 2 nodules at the base of the petiole as early as 48 h after culture. All of these structures developed into shoots during incubation.Abbreviations FAA formalin 5%–70% ethanol, 90%-acetic acid 5% - GMA glycol methacrylate - BA 6-benzyladenine     

Cell-mediated oxidation of LDL: Comparison of different cell types of the atherosclerotic lesion

The three major cell types of the human atherosclerotic lesion — macrophages (Mø), smooth muscle cells (SMC) and endothelial cells (EC) — were compared for their ability to oxidise low density lipoprotein (LDL) in vitro under identical conditions. Near-confluent cultures were incubated for up to 48 h with 50 μg protein/ml LDL in Ham's F10 medium supplemented with 7 μM Fe²⁺. All three cell types oxidised LDL readily using our culture conditions. After 24 and 48 h, the degree of LDL oxidation was in the order: Mø > SMC > EC when based on cell growth area and EC > SMC > Mø when based on cellular DNA content. However, LDL oxidation in vitro progressed more slowly between 24 and 48 h, probably due to increasing toxicity to the cells and/or depletion of polyunsaturated fatty acids. We therefore compared the time of onset of LDL oxidation. The earliest increase in LDL oxidation was always apparent with SMC. Gas chromatography revealed that LDL oxidation by all three cell types followed a similar pattern. The polyunsaturated fatty acids linoleic acid (18:2) and arachidonic acid (20:4) were depleted (to 10.3–18.1% and 4.5–24.7% respectively, compared to native LDL), whereas the content of stearic acid (18:0) and oleic acid (18:1) remained unchanged. Cholesterol was depleted (to 54.1–75.6% of native LDL) with a concomitant rise in 7β-hydroxycholesterol (to 60.6–128.1 μg/mg LDL). This corresponds to a conversion of 4.9, 9.5 and 10.4% of LDL cholesterol in EC-, SMC- and Mø-modified LDL respectively. All three cell types showed significant toxicity in the oxidising culture after 24 h. The possible relevance to LDL oxidation in atherosclerosis is discussed.     

Co-expression of saccharifying alkaline amylase and pullulanase in Micrococcus halobius OR-1 isolated from tapioca cultivar soil

Bacterial isolates from Tapioca cultivar soil were systematically identified. The effect of culture conditions and medium components on the production of extracellular amylase and pullulanase by Micrococcus halobius OR-1 were investigated. Amylase and pullulanase activity in the cell-free supernatant reached a maximum of 8.6 U/ml and 4.8 U/ml after 48 h, respectively. The enzyme converted the complex polysaccharides starch, dextrin, pullulan, amylose and amylopectin predominantly into maltotriose. Saccharification of 15% cereal, tuber starches and root starches with the whole cultured cells (WCC) or cell-free supernatant (CFS) showed comparable and complete saccharification within 90 min. These saccharifying enzymes had a pH optimum of 8.0 and were stable over a broad pH range of 6–12. Thus the coexpressed physicochemically compatible extracellular amylase and pullulanase produced by the Micrococcus halobius OR-1 strain might have important value in the enzyme-based starch saccharification industry.     

Induction of division and differentiation of somatic embryos in the leaf epidermis of Gaillardia picta

Somatic embroys and subsequent plant regeneration were obtained from isolated leaf epidermis of Gaillardia picta. Abaxial and adaxial epidermal peels (monolayer) from 45 days old aseptic seedlings were isolated and segments measuring 5 mm x 3 mm were cultured on B₅ basal medium supplemented with various growth regulators such as naphthaleneacetic acid or indolebutyric acid and benzylaminopurine or kinetin. Within 12 h of culture the epidermal cells showed receding of cytoplasm from the walls. After 48 h of incubation 3 or 4 localized zones, each consisting of 3–8 cells that accumulated cytoplasm and stained densely, were observed. Mitotic divisions occurred in these zones on day 3 of culture and localized masses of callus were observed in 95% of the cultures after 10 days. In another 5 days, the callus differentiated somatic embryos or roots, depending on the growth regulators and their concentration in the medium.Abbreviations Used BAP 6-benzylamiopurine - IBA indolebutyric acid - Kn kinetin - NAA -naphthaleneacetic acid     

Relation between lipid peroxidation and iron concentration in mouse liver after acute and subacute cadmium intoxication

In this study the effect of acute and subacute cadmium (Cd) intoxication on iron (Fe) concentration and lipid peroxidation (LPO) was investigated in the livers of Swiss mice. Animals were divided into two groups: the Cd group – mice intoxicated with Cd and controls. In acute time-response studies, Fe and malondialdehyde (MDA) levels were determined at 4, 6, 12, 24 and 48 h after a single oral dose of Cd (20 mg Cd/kg b.w.). In the subacute experiment, mice were given 10 mg Cd/kg b.w. orally every day for 14 days; Fe and MDA contents were determined in liver after 1 and 2 weeks. Acute Cd intoxication induced a significantly increased hepatic Fe content after 4 and 6 h, and a statistically significant increase in MDA 6, 12 and 24 h after Cd administration, although a significantly decreased MDA level was observed after 48 h. The results suggest development of early oxidative stress in livers of mice after acute intoxication with Cd. The decreased MDA observed after 48 h occurred presumably due to the adaptive response of the organism. Subacute Cd intoxication induced a significant decrease of hepatic Fe and MDA levels at both investigated time intervals compared with control. These results indicate a positive correlation between hepatic Fe and MDA content and suggest that prolonged Cd intoxication decreases hepatic LPO indirectly, by reducing the Fe content of mouse liver.