Probing the enantioselectivity of a diverse group of purified cobalt-centred nitrile hydratases.

In this study a diverse range of purified cobalt containing nitrile hydratases (NHases, EC 4.2.1.84) from Rhodopseudomonas palustris HaA2 (HaA2), Rhodopseudomonas palustris CGA009 (009), Sinorhizobium meliloti 1021 (1021), and Nitriliruptor alkaliphilus (iso2), were screened for the first time for their enantioselectivity towards a broad range of chiral nitriles. Enantiomeric ratios of >100 were found for the NHases from HaA2 and CGA009 on 2-phenylpropionitrile. In contrast, the Fe-containing NHase from the well-characterized Rhodococcus erythropolis AJ270 (AJ270) was practically aselective with a range of different α-phenylacetonitriles. In general, at least one bulky group in close proximity to the α-position of the chiral nitriles seemed to be necessary for enantioselectivity with all NHases tested. Nitrile groups attached to a quaternary carbon atom were only reluctantly accepted and showed no selectivity. Enantiomeric ratios of 80 and >100 for AJ270 and iso2, respectively, were found for the pharmaceutical intermediate naproxennitrile, and 3-(1-cyanoethyl)benzoic acid was hydrated to the corresponding amide by iso2 with an enantiomeric ratio of >100.

Context effects on the neural correlates of recognition memory: an electrophysiological study.

Event-related potentials (ERPs) were recorded during a recognition memory test for previously studied visual objects. Some studied objects were paired with the same context (landscape scenes) as at study, some were superimposed on a different studied context, and some were paired with new contexts. Unstudied objects were paired with either a studied or a new context. Three ERP memory effects were observed: an early effect elicited by all stimuli containing at least one studied component; a second effect elicited only by stimuli in which both object and context had been studied; and a third effect elicited by stimuli containing a studied object. Thus, test stimuli engaged three distinct kinds of memory-related neural activity which differed in their specificity for task-relevant features.

When more means less: neural activity related to unsuccessful memory encoding.

The neural correlates of memory encoding have been studied by contrasting neural activity elicited by items at the time of learning according to whether they were later remembered or forgotten [1]. Previous studies have focused on regions where neural activity is greater for subsequently remembered items [2-8]. Here, we describe regions where activity is greater for subsequently forgotten items. In two experiments that employed the same incidental learning task, activity in an overlapping set of cortical regions (posterior cingulate, inferior and medial parietal, and dorsolateral prefrontal) was associated with failure on a subsequent memory test.

Abdominal fat distribution differently affects muscle strength of the upper and lower extremities in women.

BACKGROUND/OBJECTIVES: In obese subjects, reduced muscle mass and strength are associated with impaired functional and metabolic capacity. We therefore investigated the association between abdominal fat distribution, fat mass index and maximum muscle strength of upper and lower extremities in obese patients. SUBJECTS/METHODS: One hundred and fifty five outpatients with a body mass index (BMI)⩾30 kg/m2 (105 women; 45.1±14.6 years; BMI 43.5±8.2 kg/m2) were included in the study. Grip and knee extension strength were measured with dynamometers. Using bioelectric impedance analysis, fat-free mass and fat mass indices (FFMI, FMI) were calculated. The influence of age, weight, FFMI, FMI and waist-to-hip ratio (WHR) on grip and knee extension strength was investigated in a risk factor-adjusted regression analysis. RESULTS: BMI did not correlate with hand grip or knee extension strength. The regression model confirmed a positive effect of higher weight on strength parameters; however, increased FMI was shown to have a negative effect on strength in both sexes (women, knee: ß: -1.495, confidence interval (CI): -2.075 to -0.0914, P<0.0001; women, hand: ß: -0.714, CI: -1.156 to -0.273, P=0.002; men, hand: ß:-1.448, CI: -2.618 to -0.278, P=0.016). Although increased WHR positively influenced knee extension strength (ß: 24.286, CI: 0.728-47.844, P<0.043), it did not affect grip strength in women. This association was not seen in men. CONCLUSIONS: Body fat distribution rather than BMI alone needs to be considered when evaluating strength parameters in obesity. As the relationship between obesity and strength of the upper versus lower extremities differs, grip strength cannot be considered an indicator of whole body strength in obese individuals.

Comparison of radiofrequency kyphoplasty and balloon kyphoplasty in combination with posterior fixation for the treatment of vertebral fractures.

BACKGROUND: In case of complex vertebral fractures, posterior fixation is often required for correction of deformity and instability. Fixation is commonly supported by balloon kyphoplasty (BKP) anterior. A development of BKP is radiofrequency-targeted vertebral augmentation (RF-TVA), which leads to comparable results for augmentation and pain relief. OBJECTIVE: This prospective study evaluates the outcome of posterior fixation combined with RF-TVA or BKP, respectively. METHODS: VAS, ODI, kyphosis angle and vertebral height of 44 patients were evaluated preoperatively, 3 and 12 months postoperatively. RESULTS: Both treatments improved vertebral height and kyphosis angle. At 12 months, vertebral height restoration was still significantly better in the BKP group (p < 0.001) and the improvement of kyphosis angle was comparable between both groups (p = 0.71). VAS and ODI improvements were significantly better in the RF-TVA group (p < 0.001). 8% of BKP patients had cement extravasations, compared to 10.5% in the RF-TVA group (p = 1.0). CONCLUSIONS: Combining posterior fixation with RF-TVA leads to better results of VAS and ODI, whereas the vertebral height restoration was favorable for patients treated with BKP. Cement leakage was comparable between both groups. It was asymptomatic and within reported ranges. Limitations of this study are the patient number and different stabilization instrumentation.

The 2D : 4D Digit Ratio as a Biomarker for Autism Spectrum Disorder.

It has been suggested that the second (2D, index finger) to fourth (4D, ring finger) digit ratio, 2D : 4D, may be a biomarker for the risk of developing autism. The aim of the current study was to determine the usefulness of the 2D : 4D digit ratio as biomarker for autistic traits. N = 401 healthy young volunteers participated in the study. For both hands, digit lengths were measured using digital Vernier calipers. In addition to demographics, the Autism Spectrum Quotient (AQ) questionnaire was completed, comprised of five subscales, assessing "social insights and behavior," "attention switching," "communication," "imagination," and "attention to detail." Overall, no significant correlations were observed between the AQ total score, its subscales, and the 2D : 4D digit ratio. For women, the left hand 2D : 4D digit ratio correlated significantly with the subscale score "communication" (r = -0.142; p = 0.036). For men, a significant positive correlation was found between the left 2D : 4D digit ratio and the total AQ score (r = 0.157; p = 0.042) and AQ subscale "attention switching" (r = 0.182; p = 0.017). In conclusion, gender specific associations between the 2D : 4D digit ratio and specific autism traits were observed, which were stronger in men than in women. Future studies should be conducted in patients that are formally diagnosed with autism.

Dissociable human perirhinal, hippocampal, and parahippocampal roles during verbal encoding.

The precise contribution of perirhinal cortex to human episodic memory is uncertain. Human intracranial recordings highlight a role in successful episodic memory encoding, but encoding-related perirhinal activation has not been observed with functional imaging. By adapting functional magnetic resonance imaging scanning parameters to maximize sensitivity to medial temporal lobe activity, we demonstrate that left perirhinal and hippocampal responses during word list encoding are greater for subsequently recalled than forgotten words. Although perirhinal responses predict memory for all words, successful encoding of initial words in a list, demonstrating a primacy effect, is associated with parahippocampal and anterior hippocampal activation. We conclude that perirhinal cortex and hippocampus participate in successful memory encoding. Encoding-related parahippocampal and anterior hippocampal responses for initial, remembered words most likely reflects enhanced attentional orienting to these positionally distinctive items.

A rapid micro scale method for the detection of lysopine and nopaline dehydrogenase activities.

A rapid and sensitive method has been developed to determine lysopine dehydrogenase (EC 1.5.1-) and nopaline dehydrogenase activities in crown gall tumour tissues. By this method, enzyme activities as low as 0.2 micrometerol octopine or nopaline per h per g fresh weight tumour tissue can still be detected. In non-infected young pea seedlings, no lysopine dehydrogenase activity was detected.

Properties of D(+)-lysopine dehydrogenase from crown gall tumour tissue.

D(+)-Lysopine dehydrogenase of an octopine-type Crown Gall tumour has been partially purified and a number of kinetic parameters have been determined. D(+)-Lysopine dehydrogenase catalyzes the reductive condensation of pyruvate and one of at least six different L-amino acids, as well as the reverse reactions, with preferential use of NADP(H) as a cofactor. The optimal pH for both reductive and oxidative reactions has been determined. At pH 6.8, L-lysine has of all the amino acids the lowest Km value, while at the same pH the highest V was found with L-arginine and L-histidine. The isoelectric point of D(+)-lysopine dehydrogenase is about 4.5.

Discrimination between lectins with similar specificities by ratiometric profiling of binding to glycosylated surfaces; a chemical 'tongue' approach.

Carbohydrate-lectin interactions dictate a range of signalling and recognition processes in biological systems. The exploitation of these, particularly for diagnostic applications, is complicated by the inherent promiscuity of lectins along with their low affinity for individual glycans which themselves are challenging to access (bio)synthetically. Inspired by how a 'tongue' can discriminate between hundreds of flavours using a minimal set of multiplexed sensors and a training algorithm, here individual lectins are 'profiled' based on their unique binding profile (barcode) to a range of monosaccharides. By comparing the relative binding of a panel of 5 lectins to 3 monosaccharide-coated surfaces, it was possible to generate a training algorithm that enables correct identification of lectins, even those with similar glycan preferences. This is demonstrated to be useful for discrimination between the cholera and ricin toxin lectins showing the potential of this minimalist approach for exploiting glycan complexity.

[A Comparison of Dorsal Decompression and Dorsal Decompression Combined with the Dynamic Stabilisation Device LimiFlex™]. / Vergleich dorsaler Dekompression nicht stabilisiert und dynamisch stabilisiert mit LimiFlex™.

BACKGROUND: Lumbar spinal canal stenosis is commonly treated by dorsal decompression. However, resection of posterior elements increases the biomechanical instability and may lead to further complications. In order to prevent this, fusion of the involved segments is often performed. But further complications may be associated with this, for example, highly reduced flexibility. In order to overcome fusion-related problems, dynamic stabilisation devices, like the new LimiFlex™ Paraspinous Tension Band (PSB), have been developed. This prospective study compares dorsal decompression without stabilisation and dorsal decompression with stabilisation using the PSB in patients with lumbar canal stenosis. METHODS: Sixty-three patients with stenosis involving one or two lumbar vertebral levels were treated with dorsal decompression. Forty received the PSB following decompression surgery. Back, hip and leg pain as well as patient's degree of disability were assessed preoperatively and at 3, 6 and 12 months postoperatively for all patients. Evaluations were conducted using the visual analogue scale (VAS) and the Oswestry disability index (ODI). Adverse events during the study period were evaluated. RESULTS: Patients who received the PSB experienced a better pain relief and improvement in disability compared to patients who received treatment through decompression only. A significant difference of VAS and ODI development was found between both groups when treating two vertebral levels. Furthermore, the total number of adverse events was lower in the PSB group compared to the decompression group. CONCLUSION: Dynamic stabilisation using the PSB delivers better results in terms of VAS and ODI values when compared to only dorsal decompression. In addition, it is also associated with a lower number of complications. The PSB is most favourable when 2 levels are treated.

Functional and mutated agrocinopine synthase genes on octopine T-DNAs.

Agrocinopine synthase genes have so far been detected only on the Agrobacterium tumefaciens nopaline Ti plasmid pTiC58 and on the A. rhizogenes Ri plasmid pRiA4. The TA region of the octopine/cucumopine (o/c) A. vitis Ti plasmid pTiTm4 strongly resembles the TL DNA of biotype I octopine Ti plasmids. In addition, it carries an intact and functional agrocinopine synthase gene close to its left border. TA/TL sequence comparison shows that the biotype I TL region still carries a 5'-deleted acs gene, strongly indicating that this gene was originally part of the TL region and belongs to the "common DNA" region of octopine and nopaline Ti plasmids. Tm4 and C58C1 (pTiTm4) remove agrocinopine A from the medium indicating that pTiTm4 also carries agrocinopine uptake genes. In spite of this, Tm4 and related strains are resistant against agrocin 84. Two o/c Ti plasmids that are closely related to pTiTm4, pTiHm1, and pTiAB3, have a mutated acs gene; whereas Hm1 can still take up agrocinopine A, AB3 cannot. These results yield new insights in the evolution of octopine, nopaline, and o/c strains.

A T-DNA from the Agrobacterium tumefaciens limited-host-range strain AB2/73 contains a single oncogene.

Agrobacterium tumefaciens strain AB2/73 isolated from Lippia canescens has been described as a limited-host-range strain. Its tumor-inducing (Ti) plasmid has been found to lack DNA homology to known T-DNAs (L. Unger, S. F. Ziegler, G. A. Huffman, V. C. Knauf, R. Peet, L. W. Moore, M. P. Gordon, and E. W. Nester. J. Bacteriol. 164:723-730, 1985). We have isolated a T-DNA from AB2/73 by using a heterologous border sequence as a probe. The AB2/73 T-DNA sequence (3,504 bp) is flanked by canonical border sequences, has no detectable DNA homology with other T-DNAs, and contains only two genes: lsn (Lippia strain nopaline synthaselike gene) and lso (Lippia strain oncogene). The lso gene induces nondifferentiating tumors on a limited number of hosts when transferred by a Ti plasmid from a wide-host-range strain. Part of the predicted Lso protein is weakly homologous to other Agrobacterium oncoproteins encoded by rolB, rolB, orf13, gene e, gene 5, and gene 3'. A 28-kb fragment corresponding to the virA to virE region was cloned by using a heterologous vir fragment as probe. The AB2/73 vir region is homologous to most of the C58 virulence region; however, the virA gene is most related to the virA gene of the Agrobacterium vitis limited-host-range strain Ag162.

Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA.

The iaaM gene from different plant-associated bacteria encodes a tryptophan monooxygenase (IaaM) that catalyzes the synthesis of indole-3-acetamide (IAM), a precursor of indole-3-acetic acid (IAA). Unlike the IaaM proteins from other bacteria, Agrobacterium spp. T-DNA-encoded IaaM proteins carry a 200 amino acid N-terminal extension with low homology to various members of the RolB protein family. This family is composed of 18 highly divergent T-DNA-encoded proteins, the basic functions of which are still largely undetermined. Deletion of the 5' rolB-like extension of the iaaM gene from Agrobacterium tumefaciens strain Ach5 did not lead to a reduction in IAM synthesis in plants. When expressed in tobacco, the rolB-like fragment did not affect growth or morphology. An iaaM homolog (A4-orf8) from the TL-DNA of Agrobacterium rhizogenes strain A4 also was investigated. Neither the full-size A4-orf8 gene nor the 5'-truncated form induced detectable IAM synthesis. Plants expressing the rolB-like part of the A4-orf8 gene, however, were dwarfed and mottled to various extents and synthesized abnormally high amounts of glucose, fructose, sucrose, and starch.

Limited host range Ti plasmids: recent origin from wide host range Ti plasmids and involvement of a novel IS element, IS868.

Agrobacterium tumefaciens biotype III octopine strains have been isolated from grapevine tumors worldwide. They comprise limited and wide host range (LHR and WHR) strains that carry related tumor-inducing (Ti) plasmids with two T-regions, TA and TB. The WHR TA-region resembles the biotype I octopine region, whereas the LHR TA-region is a recent deletion derivative of the WHR TA-region, which lacks the iaa genes and part of the ipt gene. Sequencing of the TA-region of the ubiquitous LHR strain AB3 showed that the deleted region is replaced by an insertion sequence (IS) element, IS868, which resembles the IS51 element of Pseudomonas syringae subsp. savastanoi. The Ti plasmid of LHR strain Ag57 carries essentially the same iaa gene deletion as pTiAB3, but lacks IS868. We propose that the LHR Ti plasmids arose by the recent insertion of an IS868 element into the TA-region of a WHR-type Ti plasmid, followed by transposition to a nearby site. The deletion was caused during the second transposition or by later recombination between the two IS868 copies. Biotype III octopine strains also carry an IS51-like sequence close to the TB iaa genes. Our results confirm and extend earlier observations indicating that IS51-like elements in Pseudomonas and Agrobacterium are associated with iaa genes and played a major role in Ti plasmid evolution.

Natural instability of Agrobacterium vitis Ti plasmid due to unusual duplication of a 2.3-kb DNA fragment.

The octopine/cucumopine (o/c) Ti plasmids of Agrobacterium vitis carry two T regions, TA and TB. The TA region resembles the octopine TL region. The TB region contains the auxin synthesis genes TB-iaaM and TB-iaaH and the cucumopine synthesis gene cus. Within the group of o/c isolates, strains 2608 and 2641 are closely related. However, 2641 lacks the TB region. The restriction maps of pTi2608 and pTi2641 were established and showed that the TB deletion resulted from intramolecular recombination between two directly repeated sequences separated by 66 kb in pTi2608. The 2,294-bp repeated sequence lacks inverted repeats and does not duplicate its target site, indicating that it is not a classical bacterial insertion sequence (IS element). It was therefore called an RSAv element (repeated sequence of A. vitis). The RSAv element carries two open reading frames: ORF234 is homologous to the traR gene of the A. tumefaciens nopaline Ti plasmid pTiC58; ORF488 is homologous to the sucrose phosphorylase gene of Leuconostoc mesenteroides and the glucosyl transferase A gene of Streptococcus mutans. The RSAv repeat starts precisely at the start codon of ORF488 and ends two base pairs 3' of the stop codon of ORF234. The structural organization of the RSAv element suggests that the amplification event did not result from a random amplification process. A study of the distribution of the two RSAv copies (RSAv-1 and RSAv-2) in pTi2608 and other o/c isolates indicates that the ancestor o/c Ti plasmid contained only RSAv-1 and that this sequence was duplicated at one point during the divergent evolution of the o/c Ti plasmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and distribution of tartrate utilization genes in the grapevine pathogen Agrobacterium vitis.

Agrobacterium vitis is a common pathogen of grapevine. Most strains utilize tartrate, an abundant compound in grapevine. Strain AB3 carries two tartrate utilization (or TAR) regions: TAR-I (on the large pTrAB3 plasmid) and TAR-II (on the AB3 Ti plasmid). TAR-I and TAR-II were structurally and functionally analyzed and are similar to the TAR-III region from the tartrate utilization plasmid pTrAB4 of the nopaline-type A. vitis strain AB4 (Crouzet and Otten, J. Bacteriol. 1995, 177:6518-6526). The minimal tartrate utilization region of TAR-I contains four genes (ttuA-ttuD). The ttuC gene is homologous to the tartrate dehydrogenase gene from Pseudomonas putida. Outside the minimal region a second ttuC-like gene is found (ttuC') which is transcribed and complements a ttuC mutant. Most grapevine isolates carry one or two of the three characterized TAR regions and show a considerable degree of polymorphism around these regions.

Role of T-region borders in Agrobacterium host range.

The limited host range AB3 strain of Agrobacterium tumefaciens induces tumors by transferring two T-regions, TA and TB. TA is a deleted version of the well-known biotype I octopine TL-region that lacks the iaa and ipt genes, but carries an intact oncogene, gene 6b, and typical left and right border sequences. TB carries two iaa genes that together code for the synthesis of indoleacetic acid. Gene 6b and the iaa gene act synergistically when transferred in a coinoculation experiment. The TA-region of the limited host range isolate Ag57 is related to the TA-region of AB3, but differs from it at several positions. The most significant difference is the absence of the right border region. In spite of this, Ag57 and the exconjugant strain C58C9(pTiAg57) induce normal tumors on Nicotiana rustica and Vitis vinifera. Various experiments indicate that gene 6b of the Ag57 TA-region is active and transferred in spite of the absence of the right border. On N. tabacum, C58C9(pTiAg57) is nononcogenic but becomes oncogenic when the pTiAg57 TA-region is restored by the right TA border sequence of pTiAB3. Thus, the right TA border sequence of the biotype III limited host range strains is required for tumor induction on some hosts, but not on others.

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.

Electrophysiological correlates of memory encoding are task-dependent.

Event-related potentials (ERPs) were used to investigate whether the neural correlates of successful episodic encoding differ according to the nature of the study task. At study, 16 subjects were cued to make either animacy or alphabetic decisions about visually presented words. A recognition memory test with confidence judgements followed after a delay of 30 min. For the animacy task, words that were subsequently confidently recognised were associated with a positive-going ERP modulation. By contrast, for the alphabetic task, confident recognition was associated with a negative-going ERP modulation. Both types of subsequent memory effects started shortly after word onset. These findings suggest that the neural correlates of memory encoding differ qualitatively, rather than quantitatively, according to the nature of the study task. Episodic encoding thus seems to be supported by multiple, task-specific, neural systems. The early onset of these memory effects suggests that episodic encoding can be facilitated by processes that start before the onset of the to-be-encoded item.