Influence of the Secondary Cell Wall Polymer on the Reassembly,Recrystallization, and Stability Properties of the S-Layer Protein from Bacillus stearothermophilus PV72/p2

The high-molecular-weight secondary cell wall polymer (SCWP) from Bacillus stearothermophilus PV72/p2 is mainly composed of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer. In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein. The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 μg/mg of S-layer protein. The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-l-lysine-coated electron microscopy grids. Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment. From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type. Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP. The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.Many bacteria and archaea possess crystalline bacterial cell surface layers (S-layers) as their outermost cell envelope component (3, 36, 38). S-layers are composed of identical protein or glycoprotein subunits which assemble into two-dimensional crystalline arrays showing oblique, square, or hexagonal lattice symmetry. S-layer subunits from bacteria are linked to each other and to the underlying cell envelope layer by noncovalent interactions and may therefore be isolated from whole cells or cell wall fragments by different procedures involving chaotropic agents, detergents, chelating agents, or high salt concentrations or by alkaline or acidic pH conditions. During removal of the disrupting agents, e.g., by dialysis, the S-layer subunits frequently reassemble into flat sheets or open-ended cylinders (in vitro self-assembly in suspension; for reviews, see references 37 and 38).Studies regarding the binding mechanism between the S-layer protein and the underlying cell envelope layer have shown that in gram-negative bacteria, the N-terminal region of the S-layer subunits recognizes specific lipopolysaccharides in the outer membrane (9, 29, 41). For Aeromonas hydrophila it was found, however, that the C-terminal part of the S-layer protein is essential for interaction with the outer membrane (40). A similar observation was reported for the S-layer protein from the gram-positive Corynebacterium glutamicum. A hydrophobic stretch of 21 amino acids located at the C-terminal end of the S-layer protein was found to interact with a hydrophobic layer in the cell wall proper that most probably consisted of mycolic acid (8). In earlier studies it was suggested that secondary cell wall polymers could represent the binding sites for the S-layer proteins from Bacillus sphaericus (15, 16) and Lactobacillus buchneri (24).Recently, a high-molecular-weight secondary cell wall polymer (SCWP) containing glucose and N-acetylglucosamine (GlcNAc) was extracted from peptidoglycan-containing sacculi of two Bacillus stearothermophilus wild-type strains (PV72/p6 and ATCC 12980 [10]). An SCWP of different chemical composition could be isolated from peptidoglycan-containing sacculi of an oxygen-induced variant strain from B. stearothermophilus PV72/p6 (35). The SCWP produced by this variant strain (B. stearothermophilus PV72/p2) is mainly composed of GlcNAc and N-acetylmannosamine (ManNAc) and shows a molecular weight of about 24,000 (33). Binding studies with proteolytic cleavage fragments and native peptidoglycan-containing sacculi revealed that the N-terminal region is involved in anchoring the S-layer subunits to the rigid cell wall layer (10, 11, 33). Several observations have supported the notion that a specific recognition and binding mechanism exists between the SCWP and the N-terminal region of the S-layer proteins from B. stearothermophilus strains. (i) Despite the overall heterogeneity, S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region and are capable of binding to an SCWP of identical chemical composition. (ii) B. stearothermophilus PV72/p6 and the oxygen-induced p2 variant produce an SCWP of different chemical composition and structure. (iii) The S-layer protein from B. stearothermophilus PV72/p2 did not recognize native peptidoglycan-containing sacculi from B. stearothermophilus wild-type strains as binding sites (35). (iv) The S-layer protein from B. stearothermophilus PV72/p6 (SbsA) and the oxygen-induced p2 variant (SbsB) are encoded by different genes which show little overall identity (19, 20), and only SbsB possesses a typical S-layer homologous (SLH) domain (23) at the N-terminal part.By sequence comparison, SLH domains (23) were identified on the N-terminal part of several S-layer proteins (6, 13, 23, 27, 30) or at the very C-terminal end of cell-associated exoenzymes and exoproteins (21, 22, 25, 26). SLH domains were suggested to anchor these proteins permanently or transiently to the cell surface. So far, evidence for a binding function of an SLH domain was provided for the S-layer protein of Thermus thermophilus (30) and for the outer-layer proteins of the cellulosome complex from Clostridium thermocellum (21, 22).In the present study, the influence of the SCWP on the formation of self-assembly products in suspension and on the recrystallization properties of the S-layer protein from B. stearothermophilus PV72/p2 on solid supports such as poly-l-lysine-coated electron microscopy (EM) grids was investigated. Moreover, studies on the stability of the S-layer protein against endoproteinase Glu-C attack in the presence and the absence of the SCWP were carried out.     

On the pungent principle of Matricaria pubescens

The pungent principle of the aerial parts and roots of Matricaria pubescens has shown to be the known thienyl-hexadien-isobutylamide. The structure was confirmed by ¹³C NMR analysis. In addition, the roots afforded small amounts of decadien-isobutylamide, and the aerial parts afforded large amounts of herniarin. The chemosystematic significance of amide accumulation within the tribe Anthemideae is briefly discussed.     

The astacin protein family in Caenorhabditis elegans.

In the nematode Caenorhabditis elegans, 40 genes code for astacin-like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the other C. elegans astacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS-4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS-7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS-7 fluorescence becomes detectable just before hatching. In C. elegans astacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C-terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG-like, CUB, SXC, and TSP-1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS-39 shows a striking resemblance to human BMP-1 and the tolloids.     

Envisioning Power: Ideologies of Dominance and Crisis

Envisioning Power: Ideologies of Dominance and Crisis. Eric R. Wolf. Berkeley: University of California Press, 1999. 339 pp.     

Twelve Thousand Years: American Indians in Maine

Twelve Thousand Years: American Indians in Maine. Bruce J. Bourque. with Steven L. Cox and Ruth H. Whitehead. Lincoln: University of Nebraska Press, 2001.368 pp.     

Automated determination of clomipramine and its major metabolites in human and rat serum by high-performance liquid chromatography with on-line column-switching

A fully automated method including column-switching and isocratic high-performance liquid chromatography (HPLC) was developed for simultaneous determination of the tricyclic antidepressant clomipramine and its metabolites demethylclomipramine, 2-, 8-, and 10-hydroxyclomipramine, 2-, and 8-hydroxydemethylclomipramine and didemethylclomipramine in serum. After serum injection into the HPLC system and on-line sample clean-up on a clean-up column (Hypersil CN; 10×4.6 mm) by an eluent consisting of 35% acetonitrile and 65% deionized water, the chromatographic separation was performed on an analytical column (LiChrospher CN; 250×4.6 mm I.D.) by an eluent consisting of 38% acetonitrile and 62% aqueous sodium perchlorate (0.02 M, pH 2.5). The UV detector was set at 260 nm. The limit of quantification was about 15 ng/ml for all analytes. The coefficients of variation ranged between 3 and 12% with recovery rates between 64 and 110%. Linear regression analyses revealed coefficients of correlation between 0.98 and 0.99. The method could be applied to therapeutic drug monitoring as well as metabolism studies in man and rat.     

Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum

Conventional screening protocols for transgene integration in mice employ tail tips or blood samples as sources to obtain genomic DNA preparations. We have developed a simple alternative non-surgical method. Epithelial cells are scraped off the inner surface of the rectum with a sterile plastic inoculation loop and are lysed with Kawasaki buffer. The lysate can be directly examined in a polymerase chain reaction (PCR) analysis without any need for further DNA purification. This procedure causes minimal harm and stress to the animals and repeated samples can be obtained as often as necessary. This technique has been used successfully to identify transgenic mice from a number of different lines. The method allows quick screening of numerous animals and contributes to a reduction of the number of surgical biopsies required     

Carbohydrate Structures of β-Trace Protein from Human Cerebrospinal Fluid: Evidence for "Brain-Type"N-Glycosylation

Abstract: The carbohydrate structures of β-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn²⁹ and Asn⁵⁶). Enzymatically released N -glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N -acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N -acetylglucosamine residues of nearly all structures were found to be α1,6-fucosylated. Peripheral fucose (about 20%α1,3-linked to N -acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N -acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N -acetylglucosamine only were present. At least 20 different N -glycans were identified. Analysis of the site-specific glycosylation patterns at Asn²⁹ and Asn⁵⁶ revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N -acetylglucosamine, as well as terminal N -acetylglucosamine and galactose residues, and significant amounts of N -acetylneuraminic acid in α2,3 linkage), this protein can be classified as "brain-type" glycosylated.     

Telomeric repeat sequences

Chromosomes not only carry transcribed genes and their regulatory DNA sequences, but also contain regions that are required for the stability and maintenace of the chromosome as a unit. These include centromeres, telomeres and origins of replication. It is clear for replication origins and centromeres that the positions of these chromosomal organelles are determined by sites of the appropriate DNA sequences, but also that functional performance requires one or more contributing proteins. Telomeres are also structurally complex, with one or more DNA components, including simple telomeric repeats and more complex telomere-associated sequences, as well as one or more specific proteins that recognize these sequences. Accumulating evidence suggests that the simple telomeric repeats are required in most, but not all species, although they are not sufficient to determine the chromosomal position of a telomere.