Diffusion tensor imaging and tractography of the white matter in normal aging: The rate-of-change differs between segments within tracts.

Knowledge concerning the normal aging of cerebral white matter will improve our understanding of abnormal changes in neurodegenerative diseases. The microstructural basis of white matter maturation and aging can be investigated using diffusion tensor imaging (DTI). Generally, diffusion anisotropy increases during childhood and adolescence followed by a decline in middle age. However, this process is subject to spatial variations between tracts. The aim of this study was to investigate to what extent age-related variations also occur within tracts. DTI parameters were compared between segments of two white matter tracts, the cingulate bundle (CB) and the inferior fronto-occipital fasciculus (IFO), in 257 healthy individuals between 13 and 84years of age. Segments of the CB and the IFO were extracted and parameters for each segment were averaged across the hemispheres. The data was analysed as a function of age. Results show that age-related changes differ both between and within individual tracts. Different age trajectories were observed in all segments of the analysed tracts for all DTI parameters. In conclusion, aging does not affect white matter tracts uniformly but is regionally specific; both between and within white matter tracts.

Cerebral atrophy as predictor of cognitive function in old, community-dwelling individuals.

OBJECTIVES: The impact of cortical and subcortical atrophy on cognitive function was examined in a sample of older community-dwelling men and women. MATERIAL AND METHODS: Magnetic resonance imaging was performed on a sample of 129 individuals [age: 68.4 +/- 3.6 years (mean +/- SD), range 64-74 years, 64 women and 65 men, Mini-Mental State Examination scores above 23] to assess cortical and subcortical atrophy. Participants also performed a number of cognitive tasks, and the measures of atrophy were used to predict performance in these tasks. RESULTS: In men, frontal cortical atrophy predicted worse performance in word fluency and the Stroop test, and occipital cortical atrophy was associated with poor performance in motor speed. In women, poor performance in motor speed was associated with subcortical atrophy at the level of the caudate nucleus. CONCLUSION: Atrophy in certain areas was associated with poor performance in specific cognitive tasks, although the amount of explained variance was rather limited in this quite homogeneous sample.

Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody.

The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

Characterization of the sec1-1 and sec1-11 mutations.

Sec1 proteins are implicated in positive and negative regulation of SNARE complex formation. To better understand the function of Sec1 proteins we have identified the nature of the temperature-sensitive mutations in sec1-1 and sec1-11. The sec1-1 mutation changes a conserved glycine(443) to glutamic acid. The sec1-11 mutation changes a highly conserved arginine(432) to proline. Based on homology and the crystal structure of the mammalian nSec1p, the corresponding amino acids localize to the 3b domain of nSec1p. Compared to the wild-type Sec1p the mutant proteins are less abundant even at the permissive temperature. Thus, the R432P and G443E mutations may cause structural alterations that affect folding and make the mutant proteins more susceptible to degradation. The remaining part is sufficient for growth and protein secretion at 24 degrees C and thus is likely to be properly folded. At 37 degrees C the mutant proteins become non-functional. In pulse-chase-type experiments the newly synthesized Sec1-1 and Sec1-11 proteins decayed similarly with the wild-type protein. Thus, the non-functionality of the mutant proteins cannot be explained by denaturation-induced degradation only. It is possible that the newly synthesized mutant proteins fold slowly and are susceptible to degradation before they have managed to fold and associate with other proteins. The mutant proteins were unable to interact with the Sec1p-interacting proteins Mso1p and Sso2p in the two-hybrid assay, even at the permissive temperature. These results localize sec1-1 and sec1-11 mutations to a domain of Sec1p and suggest a mechanism by which sec1-1 and sec1-11 cells become temperature-sensitive.

Efficient enantioselective separation of drug enantiomers by immobilised antibody fragments.

There is an increasing need for methods for efficient enantioselective separation and purification of chiral drugs. Genetic engineering provides the means for generating recombinant antibodies exhibiting extremely high specificity for even small molecular mass compounds. Here, recombinant antibody fragments have been generated for the drug diarylalkyltriazole that contains two chiral centres. Immobilised antibody fragments has been used successfully for efficient, step-wise separation of two enantiomers of the drug. Owing to the antibody specificity, one enantiomer came out in the flow-through, while the bound enantiomer could be specifically eluted. One of the antibodies tolerated solvents required both for dissolving the target molecules and for their elution for extended times and was shown to function over multiple cycles of the separation process.

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose.

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-beta-d endoglucanase, EC) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD(CBH1) and CBD(CBH2)) (1,4-beta-d glucan-cellobiohydrolase, EC), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD(Cex)) (beta-1,4-d glucanase, EC), and from C. thermocellum CipA (CBD(CipA)). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD(CBH1) < CBD(CBH2) approximately CBD(Cex) < CBD(CipA). In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBD(CipA), whose sites appeared to overlap all of the other sites.

Cross-modality priming for individual words in memory for coherent texts.

This study explores implicit memory within the domain of text processing. Three experiments were designed to study cross-modality priming in a word-stem completion test following presentation of target words in the context of a coherent text. Four main results emerged. First, we found a significant priming effect for words previously studied in a text, this priming is higher with low-frequency words than with high-frequency words. Second, subjects demonstrated more repetition priming when study and test modalities matched than when they were different. Third, the magnitude of the priming effect in the visual condition varied with the perceptual processing of the text read. Fourth, priming effects did not depend on subjects' remembering of the words of text read as measured by a yes/no recognition test since no modality effect was found in this latter memory test. These results challenge Levy's (1993) view and are discussed in the framework of the transfer-appropriate processing view proposed by Roediger, Weldon and Challis (1989).

SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast.

The exocyst is an essential multiprotein complex mediating polarized secretion in yeast. Here we describe a gene, SEM1, that can multicopy-suppress exocyst mutants sec3-2, sec8-9, sec10-2, and sec15-1. SEM1 is highly conserved among eukaryotic species. Its human homologue, DSS1, has been suggested as a candidate gene for the split hand/split foot malformation disorder. SEM1 is not an essential gene. However, its deletion rescued growth of the temperature-sensitive exocyst mutants sec3-2, sec8-9, sec10-1, and sec15-1 at the restrictive temperature. Cell fractionation showed that Sem1p is mainly cytosolic but also associates with the microsomal fraction. In linear sucrose gradients, Sem1p cosedimented with the exocyst component Sec8p. In diploid cells that normally do not form pseudohyphae (S288C background), deletion of SEM1 triggered pseudohyphal growth. This phenotype was abolished after reintroduction of either SEM1 or the mouse homologue Dss1 into the cells. In diploids that have normal capacity for pseudohyphal growth (Sigma1278b background), deletion of SEM1 enhanced filamentous growth. The functionality of both SEM1 and Dss1 in a differentiation process in yeast suggests that Dss1 indeed could be the gene affected in the split hand/split foot malformation disorder. These results characterize SEM1 as a regulator of both exocyst function and pseudohyphal differentiation and suggest a unique link between these two cellular functions in yeast.

Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs.

BACKGROUND: We have previously reported specificity improvement of an anti-testosterone monoclonal antibody (3-C4F5) by random mutagenesis of the third complementarity determining regions (CDR3s) and by phage display selection. OBJECTIVES: Here we extend the mutagenesis strategy to the other CDRs and select the mutant libraries using two different approaches in order to further fine-tune the binding properties of this recombinant Fab fragment. STUDY DESIGN: To improve the affinity the new mutant libraries were selected by using limiting, decreasing concentrations of biotinylated testosterone (TES) in solution and capturing the binders on streptavidin-coated microtiter plate. The specificity was improved by preincubating the mutant libraries in solution with a high concentration of the most problematic cross-reacting steroid, dehydroepiandrosterone sulfate (DHEAS). RESULTS: In two different light chain CDR1 mutant clones isolated from the affinity pannings, the relative TES affinity was increased over 10-fold while the cross-reactivities to related steroids were preserved at the same level as in the parental combined CDR3 mutant clone. New heavy chain CDR1 and light chain CDR2 mutants showing slightly decreased cross-reactivities were isolated from specificity selections. By combining compatible mutant CDRs together we were able to create a Fab fragment with over 12-fold higher relative TES affinity and significantly lower cross-reactivity to DHEAS when compared to the original monoclonal antibody 3-C4F5. CONCLUSIONS: Our results demonstrate that a high-affinity and selective recombinant Fab fragment working over a wide TES concentration range with clinical samples could be generated by CDR mutagenesis and phage display selection.

Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection.

The monoclonal antibodies so far developed by hybridoma technology have not had high enough specificity or affinity to distinguish the closely related steroid hormones in routine clinical assays. We have employed random mutagenesis and phage display approaches to improve the specificity of one anti-testosterone monoclonal antibody (3-C4F5). The affinity of the antibody is 0.3 x 10(9) M(-1) and the cross-reactivities with most of the related steroids are low. However, the antibody cross-reacts about 1% with dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum concentration this is about 1000-fold too high for clinical immunoassays. The complementarity-determining regions (CDRs) of the heavy and light chains, which were predicted by molecular modelling to be in close contact with the testosterone (TES) ligand, were randomized and mutant Fab libraries were cloned into a phagemid vector. Binders were selected by a competitive panning procedure. By combining the identified light and heavy chain CDRIII mutations the TES affinity was preserved at the wild-type level but DHEAS cross-reactivity was decreased to 0.03%. An important finding was that by the competitive panning procedure the overall binding specificity of the 3-C4F5 antibody was refined, since the cross-reactivities to related steroids were also significantly decreased in the combined mutant.

Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery.

Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins. Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus alpha-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted alpha-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the alpha-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiently obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast.

Acetyl xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding domain.

The axe1 gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axe1 codes for the two forms, pI 7 and pI 6.8, of acetyl xylan esterase previously characterized. The axe1 encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axe1 product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.

Yeast protein translocation complex: isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61 beta subunit.

A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion-defective sec15-1 mutation. This gene encodes a protein homologous to the beta subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane-spanning region at the C-terminus but no N-terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61 beta subunit and 34% identity to the Arabidopsis thaliana Sec61 beta subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61-2 and sec61-3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N-terminus exposed to the cytoplasm. The in vitro translated Seb1p was post-translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature-sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis.

Properties of a single-chain antibody containing different linker peptides.

Single-chain antibodies were constructed using six different linker peptides to join the VH and VL domains of an anti-2-phenyloxazolone (Ox) antibody. Four of the linker peptides originated from the interdomain linker region of the fungal cellulase CBHI and consisted of 28, 11, six and two amino acid residues. The two other linker peptides used were the (GGGGS)3 linker with 15 amino acid residues and a modified IgG2b hinge peptide with 22 residues. Proteolytic stability and Ox binding properties of the six different scFv derivatives produced in Escherichia coli were investigated and compared with those of the corresponding Fv fragment containing no joining peptide between the V domains. The hapten binding properties of different antibody fragments were studied by ELISA and BIAcoreTM. The interdomain linker peptide improved the hapten binding properties of the antibody fragment when compared with Fv fragment, but slightly increased its susceptibility to proteases. Single-chain antibodies with short CBHI linkers of 11, six and two residues had a tendency to form multimers which led to a higher apparent affinity. The fragments with linkers longer than 11 residues remained monomeric.

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment.

Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells.

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.

Quantification of hepatitis B virus DNA by competitive amplification and hybridization on microplates.

Present methods for quantification of hepatitis B virus (HBV) particles from serum samples are not sensitive enough for some recent clinical applications. We describe a test that allows quantification of HBV DNA in a broad dynamic range from less than 40 to 10(6) molecules based on competitive PCR. The specimen DNA and a known amount of an internal standard (IS) are co-amplified in the same tube with the same primers, one of which is biotinylated. The two biotinylated products can be quantified by hybridization on microplates coated with streptavidin, because their internal sequences are nonhomologous. An adequate standard curve is obtained by amplifying HBV DNA from a plasmid clone together with an IS. The ratio of amplified HBV DNA to IS DNA enables quantification of the original amount of HBV without tedious titrations of each sample with competitor. The lower limit for quantitative analysis with radioactive probes was between 4 and 40 virus particles in a 10-microliters serum samples.

Quantification of mitochondrial DNA carrying the tRNA(8344Lys) point mutation in myoclonus epilepsy and ragged-red-fiber disease.

Myoclonus epilepsy and ragged-red-fiber syndrome (MERRF) is caused by a point mutation at nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA. We analyzed leukocyte DNA from nine members of a large MERRF family using a new technique, solid-phase minisequencing. Quantitative analysis of the tRNA(8344Lys) mutation showed that the mutated mtDNA comprised from 9 to 72% of the total mtDNA in the leukocytes of these individuals. The minisequencing method is a promising tool for the diagnosis of MERRF. In addition to the identification of the tRNA(8344Lys) mutation, the relative amount of mutated mtDNA can be simultaneously determined in the same assay from one blood sample.

N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase minisequencing.

Mutations in the N-ras gene are found in one-third of patients with acute myeloid leukemia. The N-ras mutations could serve as markers for residual cells, if a highly sensitive method for detecting the mutations was available. We applied a new method, solid-phase minisequencing, to analyze bone-marrow cells from 16 patients with acute myeloid leukemia for mutations in codon 12, 13 and 61 of the N-ras gene. In the solid-phase minisequencing technique the mutations are identified by a primer extension reaction, in which a single labelled nucleoside triphosphate is incorporated into an immobilized DNA fragment previously amplified by the polymerase chain reaction. We identified N-ras mutations in 5 of the patients (30%). In one patient, we observed 2 mutations that were shown to be located in different alleles. With the solid-phase minisequencing method, we were able to determine the proportion of mutated cells in the samples. We found that in 4 of the samples only a fraction (7-64%) of the blasts carried an N-ras mutation, and in one sample practically all blast cells were mutated. The method was highly sensitive, allowing us to identify N-ras mutations even when the sample consisted of 99.7% normal cells and only 0.3% mutated blasts.