Assembly of Cellulose Synthesizing Complexes on the Plasma Membrane of Boodlea coacta

The assembly of cellulose synthesizing complexes (terminal complexes,TCs) on the plasma membrane of Boodlea coacta was investigatedduring the formation of both the matrix-rich layer (MRL) andfibril-rich layers (FRLs) of cell walls. The TCs appeared tobe located mostly within the outer leaflet of the plasma membrane,and were observed as elliptical protrusions consisting of manyparticles of about 9 nm in diameter. Their length varied from100 to 500 nm (average, 220 nm) during MRL formation and from100 to 860 nm (average, 360 nm) during FRL formation. A correlationwas found between the length of TCs and the microfibril widthin both MRL and FRL. On the E-face of the plasma membrane, numerous round protrusions(30–130 nm in diameter), consisting of many particles,8–10 nm in diameter, were also present. Their densitywas greater during FRL formation than during MRL formation.Some of these structures larger than 100 nm were associatedwith microfibril impressions and some appeared to be bound tothe TCs. These protrusions increased in number with Calcofluortreatment but decreased in number when the dye was removed fromthe culture medium. Thus, the TCs may be assembled from massesof particles aggregated on the outer surface of the plasma membrane,and may grow longer by incorporation of these masses. The appearanceof the longer TCs during FRL formation is probably due to thegreater density of these masses. (Received May 1, 1985; Accepted August 16, 1985)     

Distribution and Local Activity of Particle Complexes Synthesizing Cellulose Microfibrils in the Plasma Membrane of Closterium acerosum (Schrank) Ehrenberg

At the early stage of expansion in Closterium acerosum, cellulose-synthesizingcomplexes are distributed only in new semicells. At the laterstage, when cellulose synthesis remains restricted, many complexesenter the old semicells from the new ones. This suggests thatthe complexes in old semicells become inactivated. (Received October 13, 1982; Accepted March 3, 1983)     

纤维素降解的菌株筛选及其运用

目的:筛选降解稻草纤维素菌株,为纤维素的高效降解提供理论依据.方法:采用羧甲基纤维素钠刚果红培养基与滤纸条培养基从采集的腐木、腐土和腐叶等样品中筛选出纤维素降解菌.然后经液态发酵后测定其羧甲基纤维素酶活力与降解稻草的天然纤维素酶活力,综合考虑这两种酶活力,对其进行单独与混合发酵培养.筛选分解稻草能力较强的菌株组合.结果:初筛到5株真菌和5株细菌纤维素降解力较优的菌株.经酶活力测定后,得到分解纤维素能力较强的两株真菌F3和F5与两株细菌B1和B5,其中F3和B1的羧甲基纤维素酶活分别为705.6U、214.6U;F5和B5天然纤维素酶活分别为466.5U、204.8 U.混合培养在一定程度上能提高纤维素酶活,F3/F5具有稳定而较高的酶活力,某时间段酶活高达646.8U,且后续酶活力也保持在较高水平.而F3/B5在某时间段的酶活高达788.6U.结论:菌株的混合培养可以提高纤维素酶活.     

Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

Appearance of Chlorophyll-Protein Complexes in Greening Barley Seedlings

The sequential appearance of chlorophyll-protein complexes (CP)in greening barley leaves was studied by an improved methodof SDS-polyacrylamide gel electrophoresis (PAGE). Solubilizedthylakoid membranes were purified using a sucrose step gradientand CPs were separated by PAGE with low concentrations of SDSin solubilizing and reservoir buffers. At 10 min after the onsetof illumination, a chlorophyll-protein complex (CPX) was detected.It was a labile CP, its chlorophyll (Chl) being easily releasedfrom the apoprotein during electrophoresis. The P700-chlorophylla/b-protein complex (CPl) appeared after 45–60 min ofillumination together with P700 activity. Light-harvesting chlorophylla/b-protein complex (LHCP) began to accumulate at 2.5 h withthe beginning of Chl b synthesis. In some cases a small amountof CPa could be detected after 6 h of greening. The time-differencespectrum between homogenates of leaves illuminated for 30 and60 min had an absorbance maximum at 677 nm, showing that a redshift indicative of CPl formation began soon after completionof the Shibata shift. The time-difference spectrum between 3.5-hand 4.0-h illuminated leaves resembled the absolute spectrumof fully greened leaves, indicating that at this stage, spectralcomponents were being synthesized at the same ratio at whichthey exist in fully greened tissues. Both absolute and time-differencespectral data supported the SDS-PAGE results. (Received February 27, 1985; Accepted May 8, 1985)     

Isolation and Characterization of the Strains Producing Bacterial Cellulose

Microbiology - Two new strains producing bacterial cellulose were isolated from the Kombucha and Tibicos symbiotic communities. Based on the 16S rRNA gene analysis and the morphological,...     

Macromolecular Complexes of Xyloglucan and Cellulose Obtained by Annealing

Macromolecular complexes composed of xyloglucan and cellulosewere produced by heating amorphous celluloses with xyloglucanin water at temperatures above 160°C. The extraction ofxyloglucan from the annealed specimens required concentratedalkali which might cause microfibrils to swell. Annealed specimensobtained by heating at 200°C had a somewhat fiber-like appearanceeven though mixtures of amorphous celluloses and xyloglucanwere completely amorphous before annealing. Annealing occursspecifically between amorphous celluloses at high temperatures,where xyloglucan may be entrapped into the bundles of cellulosefibers during fiber formation rather than bound to the surfaceof the fibers. (Received September 3, 1993; Accepted December 1, 1993)     

Biological and Serological Properties of Strains of Barley Mild Mosaic Virus

The biological and serological properties of two Japanese barley mild mosaic virus (BaMMV) strains (BaMMV-Kal and BaMMV-Nal) and a German BaMMV strain (BaMMV-M) were compared. Mechanical inoculation experiments showed that these three strains differed from one another in their ability to infect specific barley cultivars. BaMMV-Kal and BaMMV-M caused similar symptoms, but BaMMV-Nal clearly differed from them in its symptoms on some barley cultivars. The three BaMMV strains efficiently infected barley plants at 15°C, whereas at 20°C BaMMV-Kal and BaMMV-M also infected many plants but BaMMV-Nal infected only a few. BaMMV-Kal and BaMMV-M were indistinguishable by ELISA, while BaMMV-Nal was distinguished from both. The biological and serological variability reported shows that BaMMV occurs as two distinct strains in Japan.     

Distribution of Acidic- and Neutral Peptidases in Germinating Barley

Activities of barley peptide hydrolases A (acidic peptidase) and H (neutral peptidase) were examined in the growing embryo and in the embryoless tissues of Himalaya barley incubated for 70 hours. Tissues examined were roots, shoots, embryo residue (scutellum + seutellar node + coleorhiza) and the embryoless portion of the kernel. Activity of the hydrolase A increased mainly in the embryo residue during 0 to 30 hours but little thereafter. Activity of hydrolase B increased mainly in the embryo residue but did so linearly over the 70 hour period. A small linear increase occurrence in the embryoless portion during this lime.     

Variety-Specific Actinomycete Complexes Associated with Barley Roots in Soddy Podzolic Soil

The root-colonizing actinomycete complexes of genotypically different barley plants grown in soddy podzolic soil were found to contain streptomycetes of the sections Cinereus (series Chromogenes, Achromogenes, and Aureus) and Roseus (series Fradiae), dominant being streptomycetes of the section Cinereus ser. Chromogenes. The abundance and diversity of soil streptomycetes in the barley rhizoplane increased in the order: var. 999-93 < var. Kumir < var. Novichok < var. 889-93. Experiments revealed functional specificity in the root-associated actinomycete complexes of different barley varieties. The actinomycete complex colonizing the barley var. 999-93 roots was distinguished by a wide range of utilizable root exudate metabolites and a low occurrence rate of antagonistic species.     

Topographic Distribution of Enzymes Synthesizing Phosphatidylcholine and Phosphatidylethanolamine in Chicken Brain Microsomes

Abstract: The localization of phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes within the transverse plane of chicken brain microsomes was investigated by using proteases (trypsin and pronase) and neuraminidase. Treatment of intact microsomes with the proteases inactivated the phosphocholine transferase completely and the ethanolamine phosphotransferase only slightly. This latter enzyme was, however, completely inactivated when deoxycholate-treated microsomes were exposed to proteases. Treatment of intact microsomes with neuraminidase had no effect on both phosphotransferases, although 65% of the sialic acid of sialoglycoproteins and 37% of that of gangliosides were removed. With deoxycholate-disrupted microsomes nearly all sialic acid from the sialoglycoproteins and about 70% of that of gangliosides were released. In parallel, the phosphoethanolamine transferase was 90% inactivated. It is suggested that phosphocholine transferase is localized on the outer face of the microsomal vesicle, whereas the phosphoethanolamine transferase could be a sialoglycoprotein, possibly situated on the inner face of the vesicle, or perhaps a transmembrane protein.     

Selective Isolation and Distribution of Sporichthya Strains in Soil

A simplified enrichment method in which centrifugation is used for selective isolation of Sporichthya strains from soil is described. Gellan gum plus 2 mM CaCl₂ stimulated growth of Sporichthya polymorpha KCC A0089 so that this organism was readily recognized on an isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27°C for 60 min and centrifuging it at 1,000 × g for 10 min. Dry heat treatment at 80°C for 60 min increased the ratio of Sporichthya colonies to nonfilamentous bacteria on a gellan gum plate. Since S. polymorpha was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating Sporichthya strains. Our isolates were identified as Sporichthya strains on the basis of their morphological and chemotaxonomic characteristics. By combining the techniques described above, we isolated a number of Sporichthya strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States. Sporichthya strains are widely distributed in the world. To our knowledge, this is the first time that Sporichthya strains have been isolated from locations other than the United States or Japan.     

The Spatial Distribution of Sucrose Synthase Isozymes in Barley

The sucrose (Suc) synthase enzyme purified from barley (Hordeum vulgare L.) roots is a homotetramer that is composed of 90-kD type 1 Suc synthase (SS1) subunits. Km values for Suc and UDP were 30 mM and 5 [mu]M, respectively. This enzyme can also utilize ADP at 25% of the UDP rate. Anti-SS1 polyclonal antibodies, which recognized both SS1 and type 2 Suc synthase (SS2) (88-kD) subunits, and antibodies raised against a synthetic peptide, LANGSTDNNFV, which were specific for SS2, were used to study the spatial distribution of these subunits by immunoblot analysis and immunolocalization. Both SS1 and SS2 were abundantly expressed in endosperm, where they polymerize to form the five possible homo- and heterotetramers. Only SS1 homotetramers were detected in young leaves, where they appeared exclusively in phloem cells, and in roots, where expression was associated with cap cells and the vascular bundle. In the seed both SS1 and SS2 were present in endosperm, but only SS1 was apparent in the chalazal region, the nucellar projection, and the vascular bundle. The physiological implications for the difference in expression patterns observed are discussed with respect to the maize (Zea mays L.) model.     

Molecular Size and Chain Length Distribution in Acanthamoeba Cellulose*

SYNOPSIS Evidence obtained by total hydrolysis, partial acetolysis, periodate oxidation, as well as treatment with amylase, emulsin, and Trichoderma virideβ-(1→4)-glucanase, verified that the alkali insoluble component of Acanthamoeba castellanii was pure β-(1→4)-glucan. The weight average chain length of the cellulose varied from DP = 3170 to DP = 4130 (mean DP = 3480) with polysaccharide obtained from seven seemingly identical cultures. Isolation of the cyst-wall cellulose by nondegrading means indicated that alkali extraction was not depolymerizing the polysaccharide. Fractionation of cellulose obtained from a single culture produced fractions from DP = 550 to DP = 4550 (mean DP = 3280; 98.7% of the original cellulose), indicating that the cellulose is polydisperse.     

纤维素在不同介质中的吸附碱的研究

文中研究了纤维素在不同介质(水、乙醇、吡啶及N,N-二甲基乙酰胺)中吸附碱测定情况并通过对比总结出了纤维素对氢氧化钠随用碱量及介质变化规律:纤维素的吸附碱量随着介质及用碱量不同而有所变化,用水作碱化介质比用有机溶剂/水作碱化介质时的吸附碱量少;在用有机溶剂/水作碱化介质时,纤维素对碱吸附表现出大致相同的变化趋势。     

Comparison of beta-Glucosidase Activities in Different Streptomyces Strains

Cellobiase (beta-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of beta-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the beta-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.     

Myelin Galactolipid Synthesis in Different Strains of Mice

Previous studies have indicated that the brains of DBA/2J (D2) mice have a more heavily myelinated CNS than those of C57BL/6J (B6) at postnatal days 17-21. However, the amount of myelin in the brains of F1 (B6 X D2) hybrids is even higher than in their parental strains. To investigate further factors involved in regulating myelinogenesis in these mice, we have focused on the synthesis of cerebrosides and sulfatides, galactolipids enriched in myelin. Brain slices from 14-, 17-, and 21-day-old D2, B6, and F1 mice were incubated with [3H]galactose and [35S]sulfate. After incubation, microsomes, myelin, and oligodendroglial cells were isolated, and the galactolipids were analyzed. At 21 days of age, the labeling of cerebrosides in F1 mice was higher than in D2 and B6 mice when the results were expressed as microsomal or myelin radioactivity per gram wet weight. At 14 and 17 days of age, the labeling of cerebrosides in F1 animals was similar to that in D2 mice and was considerably higher than that in B6 mice. The labeling of sulfatides in F1 animals was significantly higher than in the B6 parent at all ages studied, whereas it remained higher than that in the D2 parent only at 17 days of age. A similar relationship among the strains was observed when the synthesis of myelin galactolipids was estimated by measuring the in vitro activity of UDP-galactose:ceramide galactosyltransferase and 3'-phosphoadenylyl sulfate:galactosylceramide 3'-sulfotransferase. The results indicate that the increased accumulation of myelin galactolipids previously reported in the F1 mice is partially due to enhanced synthetic activity.