Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.     

Fucosylated but Not Sialylated Milk Oligosaccharides Diminish Colon Motor Contractions

Human milk oligosaccharides (HMO) are being studied by different groups exploring a broad range of potential beneficial effects to the breastfed infant. Many of these effects have been attributed to a growth promotion effect on certain gut organisms such as bifidobacteria. Additionally, evidence indicates that HMO are able to directly promote positive changes in gut epithelium and immune responses under certain conditions. This study utilizes a standardized ex vivo murine colon preparation to examine the effects of sialylated, fucosylated and other HMO on gut motor contractions. Only the fucosylated molecules, 2’FL and 3’FL, decreased contractility in a concentration dependent fashion. On the basis of IC₅₀ determinations 3’FL was greater than 2 times more effective than 2’FL. The HMO 3’SL and 6’SL, lacto-N-neotetraose (LNnT), and galactooligosaccharides (GOS) elicited no effects. Lactose was used as a negative control. Fucosylation seems to underlie this functional regulation of gut contractility by oligosaccharides, and L-fucose, while it was also capable of reducing contractility, was substantially less effective than 3’FL and 2’FL. These results suggest that specific HMO are unlikely to be having these effects via bifidogenesis, but though direct action on neuronally dependent gut migrating motor complexes is likely and fucosylation is important in providing this function, we cannot conclusively shown that this is not indirectly mediated. Furthermore they support the possibility that fucosylated sugars and fucose might be useful as therapeutic or preventative adjuncts in disorders of gut motility, and possibly also have beneficial central nervous system effects.     

Novel Glycoside Hydrolases Identified by Screening a Chinese Holstein Dairy Cow Rumen-Derived Metagenome Library

One clone encoding glycoside hydrolases was identified through functional screening of a rumen bacterial artificial chromosome (BAC) library. Of the 68 open reading frames (ORFs) predicted, one ORF encodes a novel endo-β-1,4-xylanase with two catalytic domains of family GH43 and two cellulose-binding modules (CBMs) of family IV. Partial characterization showed that this endo-xylanase has a greater specific activity than a number of other xylanases over a wide temperature range at neutral pH and could be useful in some industrial applications.The ruminal microbes possess a repertoire of hydrolases, including glycosyl hydrolases and esterases, which mediate hydrolysis and subsequent fermentation of the diets (mainly cellulose, xylan, amylopectin, amylose, pectin) (10, 18). Thus, the ruminal microbiome has been recognized as a rich source of enzymes important not only for feed and animal industries but also for the bioenergy industry (20, 26). However, this enzyme source is largely untapped because the majority of ruminal microbes remain uncultured (17). Activity-based metagenomics enables direct identification of genes and enzymes of interest by screening metagenomic libraries for desired heterologous phenotypic traits expressed in a surrogate bacterial host. Collectively, previous studies have identified 12 esterases, 10 endoglucanases, two lipases, one cyclodextrinase, one polyphenol oxidase, and one unique multifunctional glycosyl hydrolase from ruminal metagenomic libraries (2, 6, 8-9, 21, 23). These studies provided further evidence that the ruminal microbiome contains a rich source of glycosyl hydrolases possessed by as-yet-uncultured microbes. The objective of this study was to examine a bacterial artificial chromosome (BAC) library constructed previously from the ruminal samples collected from Chinese Holstein cows for hydrolases.     

研究: 奶牛与水牛初乳中乳寡糖组分比较研究

乳寡糖是由乳汁中含量丰富的固体物质组成．研究结果表明,乳寡糖有提高免疫、益生元及抗感染等作用,已发现与婴儿肠道发育、神经智力发育等多方面关系密切．水牛奶是除牛奶外的第二大奶源,国际上公认其为营养含量高、口感好的优质乳制品,但目前针对水牛乳寡糖的研究多以美洲水牛为研究对象,尚无中国水牛的相关研究．本研究利用固相萃取对已脱脂和除去蛋白质的广西水牛初乳乳汁样品进行纯化,并采用苯胺 (aniline,Bn)衍生化试剂对其进行衍生化处理,通过UPLC-ESI-Q-TOF-MS液相质谱进行优化后,对水牛初乳中的寡糖组分进行测定并与牛乳进行了对比,最终测得奶牛初乳中19种及水牛初乳中的9种乳寡糖组分,并对二者的种类及含量进行比较,发现在两种初乳的乳寡糖中,中性糖二糖m/z 385.15和中性糖三糖m/z 547.21以及酸性糖m/z 635.23均为其主要寡糖成分,与其他乳寡糖相比含量相对较高．总体而言水牛初乳中的中性寡糖占比比奶牛初乳高,二者中性糖占乳寡糖总量的比例分别为88.88%和63.16%．     

Ketosis Treatment and Milk Yield in Dairy Cows Related to Milk Acetoacetate Levels

Milk yield and milk acetoacetate (M-acac) were measured weekly for the first 6 weeks of lactation in 5 herds with a ketosis problem. Ketosis treatments and the corresponding ketotest score, were also recorded. The treatment rate was highest 7–16 days after calving. Most of these early cases were associated with low ketone levels in milk, whereas the treatment ratefor cows with high ketone levels was highest 17–31 days after calving. Nearly half of the treated cows were low-ketone animals. They were classified as ketosis cases in the cow health card records, although probably suffering from other post partum disturbances in many instances. About 40 % of the cows with high ketone levels recovered spontaneously. Reduction in milk yield associated with peak M-acac levels was transient and moderate. It was concluded that health card statistics overestimates the severity of the ketosis problem in Norway.     

Differential Cell Counting in Fraction-Collected Milk from Dairy Cows

Cell con-centrations and cell populations were studied in fraction-collected milk by use of cytofluorometric methods. The results indicate differences in the condition of udders producing milk with more and less than 100 000 cells per ml, respectively     

Effects of Short-Term Over-supplementation of Copper in Milk on Hematology,Serum Proteins,Weight Gain,and Health in Dairy Calves

Thirty-six calves were used in the present study. The animals were divided equally into three groups (control, test 1, and test 2). The three groups of calves were homogeneous for parity of dams, sex, and month of birth. From 14 days of age, in the test 1 group copper as copper sulfate (Merck Co, Germany) was added to each meal of milk at a rate of 10 mg/kg of milk for 14 days and in test 2 group copper as copper sulfate was added to each meal of milk at a rate of 20 mg/kg of milk for 14 days. Blood samples were taken by jugular venipuncture using disposable syringes at 14 (before Cu supplementation), 30, 60, and 80 days of age. Anticoagulated blood was used for CBC determination. Plane tubes were used for harvesting of serum and the amounts of total serum protein, albumin, iron, and copper were measured. Calves were weighted at birth and at the end of trial (day 80) and total gain and mean daily gain were calculated. Days of treatment for ill calves were also recorded during experiment. Group (treatment) had no significant effect on the amounts of measured parameters except MCH values (p < 0.05) which were significantly lower in test 1 group than other trial groups. Age (sampling time) had significant effects on the values of most measured parameters (p < 0.05) except WBC, lymphocyte, total protein, and fibrinogen. Significant interactions between sampling time and group were not seen for any of measured parameters. No significant differences were seen for total weight gain and mean daily gain between trial groups. Chi-square test revealed no significant difference for the days of treatment between trials groups.     

猪乳中一组高分子量蛋白的多态性与乳生长因子、窝增重的关系

利用十二烷基硫酸钠-聚丙烯酰胺凝胶不连续垂直板电泳(SDS-PAGE),对162头二花脸母猪乳中一组高分子量蛋白质(HMWP)进行了检测和分型,并运用线型模型统计分析方法分析了该基因座的不同基因型与母猪的乳生长因子(IGF-1、EGF和胰岛素)、哺乳仔猪生长(20日龄窝增重)的关系.结果表明,在三种HMWP基因型中,不同基因型母猪的乳中IGF-1浓度存在显著差异,HMWP基因型为BB型和BD型的母猪,其乳IGF-1浓度均高于DD型,其中BB型显著高于DD型(P＜0.05).乳中胰岛素浓度也存在差异的趋势,但未达到显著水平(P＞0.05),BB型和BD型母猪的乳中胰岛素浓度高于DD型.HMWP基因座不同基因型的乳EGF浓度无显著差异(P＞0.05).在三种HMWP基因型中,不同基因型母猪的20日龄窝增重存在显著差异,HMWP基因座为BB型和BD型的母猪,其20日龄窝增重均高于DD型,其中BD型显著高于DD型(P＜0.05).实验结果提示,HMWP多态性可能作为一个潜在的遗传标记应用于猪的遗传育种.     

High Molecular Weight Mucin-like Glycoprotein in Bovine Milk

We purified an enzyme hydrolyzing 2-sulfo-α-L-fucopyranose pyridylaminated 2-sulfo-α-L-fucopyranosyl-(1→2)-fucose from the acetone powder of the digestive tract of a sea urchin. The enzyme was purified 307-fold with an overall recovery of 1.63% by ammonium sulfate precipitation, cation exchange chromatography, and gel filtration chromatography. The enzyme is useful for the structural analysis of sulfated fucan.     

Novel Carbohydrate-Binding Module Identified in a Ruminal Metagenomic Endoglucanase

Endoglucanase C5614-1 comprises a catalytic module (CM) and an X module (XM). The XM showed no significant homology with known carbohydrate-binding modules (CBMs). Recombinant full-length endoglucanase could bind Avicel, whereas the CM could not. The XM could bind various polysaccharides. The results demonstrated that the XM was a new CBM.Most cellulases are modular proteins that comprise two or more discrete modules, such as catalytic modules (CMs) and carbohydrate-binding modules (CBMs), each of which can function independently (9). CBMs are classified into 59 families based on their amino acid similarity in the CAZY database (http://www.cazy.org/fam/acc_CBM.html). The main functions of CBMs are to recognize and bind polysaccharides and to increase the hydrolytic activities of the enzymes against insoluble and soluble substrates (3). Endoglucanase C5614-1 (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"ACA61140","term_id":"169640132","term_text":"ACA61140"}}ACA61140), which was identified from the metagenome of the contents of buffalo rumen (5), is a modular enzyme comprising an N-terminal signal peptide (amino acids [aa] 1 to 20), a CM belonging to the glycosyl hydrolase family 5 (aa 40 to 334), and a C-terminal X module (XM) of unknown function (aa 335 to 537) (Fig. ​(Fig.1A).1A). No linker region rich in Ser/Pro/Thr was found between the CM and the XM. In this study, we aimed to ascertain the function of the XM in the endoglucanase C5614-1.Open in a separate windowFIG. 1.Endoglucanase C5614-1 and its derivatives. (A) Modular organization of C5614-1 and its truncated derivatives. Abbreviations: SP, signal peptide; GHF5, GHF5 catalytic module; X, the X module (XM). (B) SDS-PAGE of purified recombinant proteins. Protein samples were analyzed on a 10% gel. Lane 1, protein molecular mass standard (molecular masses are shown on the left); lane 2, rC5614-1; lane 3, rGHF5; lane 4, rX.The XM showed no significant homology to known CBMs. The XM shared 25% to 33% identities and 41% to 45% similarities with about 200 amino acids at the C terminus of a xylanase (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAC36862","term_id":"143974","term_text":"AAC36862"}}AAC36862) from the ruminal bacterium Prevotella ruminicola, uncultured ruminal microbial cellulases ({"type":"entrez-protein","attrs":{"text":"ABX76045","term_id":"161898163","term_text":"ABX76045"}}ABX76045, {"type":"entrez-protein","attrs":{"text":"ACA61132","term_id":"169640121","term_text":"ACA61132"}}ACA61132, {"type":"entrez-protein","attrs":{"text":"ACA61135","term_id":"169640125","term_text":"ACA61135"}}ACA61135, {"type":"entrez-protein","attrs":{"text":"ACA61137","term_id":"169640128","term_text":"ACA61137"}}ACA61137, and {"type":"entrez-protein","attrs":{"text":"ABB46200","term_id":"78522584","term_text":"ABB46200"}}ABB46200), and an uncultured bacterial bifunctional mannanase-xyloglucanase ({"type":"entrez-protein","attrs":{"text":"ADA62505","term_id":"281335881","term_text":"ADA62505"}}ADA62505). None of these homologous polypeptides was confirmed to show carbohydrate-binding activity. An alignment of XM with these homologous sequences using ClustalW (http://www.ebi.ac.uk/Tools/clustalw) is shown in Fig. ​Fig.2.2. Seven conserved aromatic amino acid residues were found in the respective sequences. Aromatic amino acid residues in CBMs play critical roles in recognizing and binding polysaccharides (4).Open in a separate windowFIG. 2.Multiple sequence alignment of the XM (203 aa) in endoglucanase C5614-1 (aa 335 to 537), with its homologous sequences. The identical and similar amino acid residues are indicated by asterisks and dots, respectively, below the alignment. The conserved aromatic amino acid residues are indicated by arrows. GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AAC36862","term_id":"143974","term_text":"AAC36862"}}AAC36862, Prevotella ruminicola xylanase (aa 376 to 584); {"type":"entrez-protein","attrs":{"text":"ABB46200","term_id":"78522584","term_text":"ABB46200"}}ABB46200, ruminal uncultured bacterium endoglycosidase precursor protein (aa 719 to 917); {"type":"entrez-protein","attrs":{"text":"ABX76045","term_id":"161898163","term_text":"ABX76045"}}ABX76045, ruminal uncultured microorganism endo-1,4-beta-d-glucanase (aa 342 to 552); {"type":"entrez-protein","attrs":{"text":"ADA62505","term_id":"281335881","term_text":"ADA62505"}}ADA62505, ruminal uncultured bacterium bifunctional mannanase-xyloglucanase (aa 721 to 919); {"type":"entrez-protein","attrs":{"text":"ACA61132","term_id":"169640121","term_text":"ACA61132"}}ACA61132, ruminal uncultured microorganism cellulase C29-2 (aa 341 to 553); {"type":"entrez-protein","attrs":{"text":"ACA61135","term_id":"169640125","term_text":"ACA61135"}}ACA61135, ruminal uncultured microorganism cellulase C35-2 (aa 337 to 552); {"type":"entrez-protein","attrs":{"text":"ACA61137","term_id":"169640128","term_text":"ACA61137"}}ACA61137, ruminal uncultured microorganism cellulase C67-1 (aa 335 to 546). The amino acid numbers in each of the parentheses above define the range of the homologous region in each sequence.A PCR-based approach was used to produce constructs expressing C5614-1 derivatives (Fig. ​(Fig.1A).1A). Plasmid C5614 (5), carrying the endoglucanase gene C5614-1, was used as a PCR template. The primer pairs used to amplify the portions of C5614-1 encoding C5614-1 amino acids 20 to 537 (recombinant C5614-1 [rC5614-1], comprising the CM and the XM), 20 to 348 (rGHF5, containing the CM only), and 349 to 537 (rX, containing the XM only) are, respectively, as follows: C5614-1F (5′CAGCCATGGAGGCACAAGATTTTGAGACTGCTACCGAA-3′) and C5614-1R (5′-GACCTCGAGTTGTGCTATGTATTTTTTGCCGTTCTGG-3′), C5614-1F and C5614-1CM (5′-GGGCTCGAGGGTTAATGTCTCAGCCAGGTCAGGCTG-3′), and C5614-1X (5′-GGGCCATGGCCAAAGCCTATCATGGCAGCGCGTTC-3′) and C5614-1R. The underlined sequences in the primers are NcoI and XhoI restriction sites.The digested PCR products were ligated into the same digested expression vector, pET-30a(+) (Novagen, Madison, WI). The recombinant plasmids were transformed into Escherichia coli Rosetta(DE3)pLysS (Novagen), and the cloned fragments were expressed as proteins with 6×His tags at both the N and C termini. The recombinant C5614-1 derivatives were purified by affinity chromatography with Cobalt immobilized metal chromatography resin (Clontech, Palo Alto, CA), according to the user manual. Each of the purified proteins produced a single band on an SDS-PAGE gel (8), and their molecular sizes were in agreement with those of the deduced polypeptides (Fig. ​(Fig.1B1B).The hydrolytic activities of rC5614-1 and rGHF5 toward carboxymethyl cellulose (CMC) were determined essentially as described by Duan et al. (5). The pH profiles of the enzymatic reactions of rC5614-1 and rGHF5 were similar, and both showed maximum activities at pH 5.0 (data not shown). However, rC5614-1 and rGHF5 showed different temperature profiles (Fig. ​(Fig.3A).3A). The rGHF5 showed narrower pH stability and lower temperature stability profiles than those of rC5614-1 (Fig. 3B and C). These results indicated that the XM is required for the stability of rC5614-1.Open in a separate windowFIG. 3.Effects of pH and temperature on the activities and stability of rC5614-1 and rGHF5. (A) Influence of temperature on the activities of rC5614-1 and rGHF5. Cellulase activity was measured at pH 5.0 in citrate-phosphate buffer at the indicated temperatures for 10 min. Values are expressed as percentages of maximal activity at 50°C and 35°C for rC5614-1 and rGHF5, respectively. (B) Influence of pH on rC5614-1 and rGHF5 stability. Purified enzyme was first incubated in citrate-phosphate buffer (pH 2.5 to 7.0), 0.1 M Tris-HCl buffer (pH 7.5 to 8.5), or 0.1 M glycine-NaOH buffer (pH 9.0 to 12) at 4°C for 24 h, and activity was measured under optimal conditions for 10 min. Values are expressed as percentages of maximal activity when the sample was incubated under pH 5.5 and pH 6.0 for rC5614-1 and rGHF5, respectively. (C) Thermal stability of recombinant rC5614-1 and rGHF5. Purified enzymes were first incubated at the indicated temperatures for 1 h; activity was then measured under optimal conditions for 10 min. Values are expressed as percentages of untreated-sample activity.The hydrolytic activities of rC5614-1 toward particle substrates, including birch wood xylan, lichenan, and acid-swollen cellulose (ASC), were about three times greater than those of rGHF5; however, rC5614-1 and rGHF5 showed similar hydrolytic activities toward soluble substrates (Table ​(Table1).1). This result indicated that the presence of the XM enhanced the hydrolytic activity of the enzyme toward insoluble substrates but not toward soluble substrates. Similar phenomena were reported for Irpex lacteus exocellulase I (7), Clostridium thermocellum Xyn10C (1), and Clostridium stercorarium Xyn10B (2).

TABLE 1.

Specific activities of rC5614-1 and rGHF5 toward various substrates

Aeromycoflora in the Central Milk Dairy of Calcutta, India

Intramural aeromycological survey was performed at the Central Milk Dairy, Calcutta, covering eight locations within the Dairyusing Burkard personal volumetric air sampler. The locations were butter cold storage (−2 °C), cold store (8 °C), packaging section (23 °C), milk processing section (24 °C), reconstituent of skimmed milk (24 °C), quality control lab (25 °C), raw milk reception (28 °C) and loading dock (26 °C). A number of fungal spores, conidia and mycelia were recorded in different rooms: the highest spore quantity was recorded in the packaging section (23 °C) and the minimum at the butter cold store (−2 °C). The dominant spores consisted of Aspergillus niger, A flavus,Cladosporium sp., Fusarium sp., Curvularia sp.,Alternaria sp., Torula sp., Myrotheciumsp., Helminthosporium sp., Periconia sp.,Nigrospora sp. and Pithomyces sp. This revised version was published online in June 2006 with corrections to the Cover Date.     

应用高通量测序技术检测与奶牛乳产量和乳质量调控相关的miRNA

本实验将中国荷斯坦牛泌乳期高乳品质奶牛（H）和泌乳期低乳品质奶牛（L）乳腺组织作为实验对象,利用高通量测序技术进行了miRNA测序,与miRNA数据库比对,获得已知miRNA,整合miREvo和mirDeep2这两个miRNA预测软件,进行新miRNA分析,通过差异表达分析筛选组间差异miRNAs,获得56个差异表达miRNA(P <0.05,FDRq <0.05)并对差异表达miRNA进行靶基因预测;利用DAVID对靶基因进行GO(Gene Ontology)和信号通路富集分析。经过对靶基因筛选,发现了4个已报道与乳蛋白、乳脂紧密相关的功能基因：CSN3、SCD、LALBA和DGAT2。靶基因聚集的生物学功能多数参与了蛋白质和脂肪代谢,乳腺发育和分化,以及免疫功能。靶基因主要富集在MAPK 信号通路、甘油磷酸脂质代谢、缺氧诱导因子1和磷脂酰肌醇3激酶 蛋白激酶B信号转导通路。结果显示,靶基因主要富集在糖类代谢、脂肪代谢、蛋白质代谢、细胞凋亡以及免疫相关通路。     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

Arabidopsis Dynamin-Like 2 That Binds Specifically to Phosphatidylinositol 4-Phosphate Assembles into a High-Molecular Weight Complex in Vivo and in Vitro

Arabadopsis dynamin-like (ADL) 2, a member of the high-molecular weight (M(r)) dynamin family found in Arabidopsis, has been shown to be targeted to the plastid. In the chloroplast, most of the ADL2 was present in the fraction containing the envelope membranes when analyzed by suborganellar fractionation. Sucrose gradient and gel filtration experiments showed that when associated with membranes, ADL2 existed as a high-M(r) complex, whereas the soluble form existed as a monomer. The recombinant ADL2 expressed in Escherichia coli was present as a high-M(r) form and showed higher GTPase activity at a low NaCl concentration, whereas ADL2 existed as a low-M(r) form with a low level of GTPase activity at a high NaCl concentration. Electron microscopy studies revealed that the purified recombinant ADL2 formed spiral-coiled structures or rings. In the presence of guanosine-5'-O-(3-thio)triphosphate, these structures were transformed into a long rod structure. In contrast, in the presence of GDP, these structures disassembled into oligomers that were shown to be tetramer with 4-fold symmetry. Finally, a lipid-binding assay revealed that recombinant ADL2 purified from E. coli bound specifically to phosphatidylinositol 4-phosphate. Together, these results demonstrated that the biochemical properties of ADL2 were very similar to those of dynamin and other related proteins. Based on this similarity, we propose that ADL2 may be involved in vesicle formation at the chloroplast envelope membrane.     

Molecular Weight Distribution of Proteins Synthesized in Single, Identified Neurons of Aplysia

Parietovisceral ganglia from Aplysia californica were incubated in medium containing leucine-³H. Single, identified nerve cell somas were isolated from the ganglia, and their proteins extracted and separated by electrophoresis on 5% SDS-polyacrylamide gels. The distribution of total or newly synthesized proteins from the single neurons was determined by staining or slicing and liquid scintillation counting of the gels. Experiments showed that: (a) a number of proteins were being synthesized in abundance in the nerve cells; (b) different, identified neurons showed reproducibly different labeling patterns in the gels; (c) cells R2 and R15, which showed different distributions of radioactivity in the gels, had similar staining patterns; and (d) there was significant incorporation into material of high (>75,000) molecular weight in most of the cells.