On the turn-inducing properties of asparagine: the structuring role of the amide side chain, from isolated model peptides to crystallized proteins.

Asparagine (Asn) is a powerful turn-inducer residue, with a large propensity to occupy the second position in the central region of ß-turns of proteins. The present work aims at investigating the role of a local anchoring between the Asn side chain and the main chain in this remarkable property. For this purpose, the H-bonding patterns of an asparagine residue in an isolated protein chain fragment forming a γ- or a ß-turn have been determined using IR/UV double resonance gas phase spectroscopy on laser-desorbed, jet-cooled short models in conjunction with relevant quantum chemistry calculations. These gas phase data provide evidence for an original double anchoring linking the Asn primary amide side chain (SC), which adopts a gauche+ rotameric form, to its main chain (MC) local environment. From both IR spectroscopic evidence (H-bond induced red shifts) and quantum chemistry, Asn SC is found to behave as a stronger H-bond acceptor than donor, resulting in stronger MC→SC H-bonds than SC→MC ones. These gas phase structural data, relevant to a hydrophobic environment, have been used as a reference to assess the anchoring taking place in high resolution crystallized proteins of the Protein Data Bank. This approach reveals that, when the SC adopts a gauche+ orientation, the stronger MC→SC bonds are preserved in many cases whereas the SC→MC bonds are always disrupted, in qualitative agreement with the gas phase ranking of these interactions. Most interestingly, when Asn occupies the second position of central part of a ß-turn (i.e., the very turn-inducer position), the MC→SC H-bonds are also disrupted and replaced by a water-mediated SC to MC anchoring. Owing to the specific features of the hydrated Asn side chain, we propose that it could be a turn precursor structure, able to facilitate turn formation in the early events of the folding process.

Genomic imbalances associated with mullerian aplasia.

BACKGROUND: Aplasia of the müllerian ducts leads to absence of the uterine corpus, uterine cervix, and upper (superior) vagina. Patients with müllerian aplasia (MA) often exhibit additional clinical features such as renal, vertebral and cardiac defects. A number of different syndromes have been associated with MA, and in most cases its aetiology remains poorly understood. OBJECTIVE AND METHODS: 14 syndromic patients with MA and 46,XX G-banded karyotype were screened for DNA copy number changes by approximately 1 Mb whole genome bacterial artificial chromosome (BAC) array based comparative genomic hybridisation (CGH). The detected alterations were validated by an independent method and further mapped by high resolution oligo-arrays. RESULTS: Submicroscopic genomic imbalances affecting the 1q21.1, 17q12, 22q11.21, and Xq21.31 chromosome regions were detected in four probands. Presence of the alterations in the normal mother of one patient suggests incomplete penetrance and/or variable expressivity. CONCLUSION: 4 of the 14 patients (29%) were found to have cryptic genomic alterations. The imbalances on 22q11.21 support recent findings by us and others that alterations in this chromosome region may result in impairment of müllerian duct development. The remaining imbalances indicate involvement of previously unknown chromosome regions in MA, and point specifically to LHX1 and KLHL4 as candidate genes.

Molecular dynamics study of the hydration of lanthanum(III) and europium(III) including many-body effects.

Lanthanides complexes are widely used as contrast agents in magnetic resonance imaging (MRI) and are involved in many fields such as organic synthesis, catalysis, and nuclear waste management. The complexation of the ion by the solvent or an organic ligand and the resulting properties (for example the relaxivity in MRI) are mainly governed by the structure and dynamics of the coordination shells. All of the MD approaches already carried out for the lanthanide(III) hydration failed due to the lack of accurate representation of many-body effects. We present the first molecular dynamics simulation including these effects that accounts for the experimental results from a structural and dynamic (water exchange rate) point of view.

Analysis of cellular adhesion by microarray expression profiling.

Microarrays of oligonucleotides or cDNAs can be used to establish the expression profiles of numerous genes in a single experiment. We have established a microarray platform to identify genes in a number of different pathological conditions, particularly those with an inflammation component. This platform utilised the output of an eosinophil sequencing project in which 1069 sequences were identified that were not represented in the public domain. An eosinophil model cell line, AML14.3D10, was used to investigate cell adhesion. The transcription profile of adhered and non-adhered AML 14.3D10 cells was shown to be both technically and biologically reproducible. A number of genes were found differentially expressed in the adhered vs. non-adhered populations. In the adhered population, the expression of these genes was restricted compared to brain, lung, kidney and especially bone marrow. However, the differentially regulated genes were not among those genes most restricted to eosinophils. We discuss the implications of transcription profiling on gene annotation and its potential utility for the identification of targets for drug intervention.

In vivo protection against S. mansoni infection by monoclonal antibodies.

A monoclonal antibody of the IgG1 class (27.21), that was previously shown to be cytotoxic in vitro to the schistosomula, conferred partial protection in vivo against challenge infection with S. mansoni. Monoclonal antibodies of the IgE class (54.10) were not effective in these experiments, but were capable of causing specific degranulation of basophilic cells upon contact with the young larva (3-6 h). The relevance of the IgE antibodies to protection observed in immunized mice is discussed.

Mineralization of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain UCR2.

Pseudomonas sp. strain UCR2 was isolated from a multi-chemostat mating experiment between a chlorobenzoate-degrader, Pseudomonas aeruginosa strain JB2, and a chlorobiphenyl-degrader, Arthrobacter sp. strain B1Barc. Strain UCR2 differed from either of the parental organisms in that it grew on both 2-chloro- and 2,5-dichlorobiphenyl with concomitant release of chloride. Phenotypic typing by the Biolog system indicated that strain UCR2 shared greater similarity with strain JB2 (88%) than strain B1Barc (3%). In DNA:DNA hybridization experiments, genomic DNA from strain UCR2 hybridized with both strain JB2 and strain B1Barc, with the former pairing yielding a much stronger signal than the latter. In contrast, no hybridization whatsoever was observed when the parental organisms strains JB2 and B1Barc were probed against each other.

Long-term impact of clozapine and psychosocial treatment on psychiatric symptoms and cognitive functioning.

OBJECTIVE: The long-term effects of a combination of clozapine and psychosocial treatment were evaluated in a sample of treatment-refractory state hospital patients with schizophrenia. METHODS: A repeated-measure design was used. Thirty-one patients with schizophrenia received both clozapine and an enhanced psychosocial treatment program. Data were collected at baseline and at one-year, two-year, and three-year follow-ups. Psychiatric symptoms, cognitive functioning, dyskinetic movements, and discharge rate were evaluated. RESULTS: Significant reductions in psychiatric symptoms and improvement in cognitive functioning were found. Differences in the pattern of reductions in positive and negative symptoms over the course of the study were noted. The majority of subjects improved sufficiently to be discharged. CONCLUSIONS: Clozapine, when combined with psychosocial treatment, is effective for treatment of patients with schizophrenia who are not responsive to other medications.

Intergeneric conjugal transfer of Escherichia coli/Methylobacterium sp. shuttle vector.

To develop a host-vector system for Methylobacterium sp. using a construct based on a small indigenous methylotrophic plasmid, the E. coli--Methylobacterium sp. shuttle vector pWUBR (12.7 kb, Apr, Tcr) was constructed by joining the E. coli plasmid pBR328 and the cryptic plasmid pWU7 (7.8 kb), isolated from the soil facultative methylotrophic bacterium, Methylobacterium sp. strain M17. Via mobilization by the pDPT51 R plasmid, belonging to the IncP-1 incompatibility group, plasmid pWUBR was transferred into the original host of cryptic plasmid pWU7, strain M17, where a competition between the introduced hybrid plasmid and the indigenous cryptic plasmid took place, and into the plasmidless Methylobacterium sp. strain R2b. The stability of pWUBR in Tcr methylotrophic transconjugants after 25 generations of growth under nonselective conditions was more than 90% in both hosts. The ability to replicate in R2b strain demonstrates that the host spectrum of pWUBR is not restricted to the original host of pWU7 and indicates the possibility to use the present system for other methylotrophs.

An efficient method for isolation of plasmid DNA from methylotrophic bacteria.

A method, suitable for the isolation of closed circular plasmid DNA from methylotrophic bacteria is described. Improvement of cell lysis was achieved by butanol extraction of cells before application of the lytic agent. Using this method, cryptic plasmids of 7.8, 14, 36 and 200 kb were purified from soil-isolated methylotrophs.

Bacterial aerobic degradation of benzene, toluene, ethylbenzene and xylene.

Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the "upper" pathway are then mineralized by either ortho- or meta-ring cleavage ("lower" pathway). BTEX are relatively water-soluble and therefore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.

Characterization of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia.

Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contaminated by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106). Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strains Pseudomonas alcaligenes KP2 and P. fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103. DNA-DNA hybridization was used to compare both strains with well-known PCB-degraders Burkholderia cepacia strain LB400 and Ralstonia eutropha strain H850. The strain KP12 employs the same meta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degrader Pseudomonas cepacia P166. Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes. We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.

Mini-Mu transposition of bacterial genes on the transmissible plasmid.

Using the pRM30 plasmid, an Aps deletion derivative of broad host range plasmid RP4 with integrated new miniMu 5 (11 kb), we followed the transfer of Escherichia coli chromosomal genes to the recipient strain. The miniMu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated miniMu 5 rather than onto the "recipient" plasmid pNH602. The plasmid DNA in recipient cells was detected by electrophoresis. One of the acquired hybrid plasmids pTB2 was analyzed genetically and by restriction endodeoxyribonuclease digestion. A structure consisting of miniMu-chromosomal segment-miniMu as a product of Mu-mediated transposition was detected.

Parental discipline and behavior problems in young children.

The relationship between parenting practices and behavior problems in very young children was investigated via the Parent Behavior Checklist (PBC; Fox, 1994). Results indicated that parents' use of verbal and corporal punishment was the strongest predictor of reported behavior problems, accounting for 20% of overall variance and 13% of unique variance. Results for parental nurturing and expectations were mixed and suggest an indirect effect. Several demographic variables were also predictive of behavior problems. Implications for Patterson's (1986) model of the development of antisocial behavior and for the use of parent assessment as a screening tool for preventive interventions are discussed.

Psychology of computer use: XLVII. Parameters of Internet use, abuse and addiction: the first 90 days of the Internet Usage Survey.

While the addictive potential of Internet usage is a topic that has attracted a great deal of attention, as yet little research has addressed this topic. Preliminary data from the Internet Usage Survey shows that most of the 563 users reported instances of Internet use interfering with other aspects of their lives, most commonly problems with managing time. A subgroup of users endorsed multiple usage-related problems, including several similar to those found in addictions. Younger users tended to have experienced more problems.

Perception and pain thresholds for cutaneous heat and cold, and rectal distension: associations and disassociations.

BACKGROUND: Hypersensitivity to somatic or visceral pain has been reported in numerous clinical conditions such as fibromyalgia or the irritable bowel syndrome, and general hypersensitivity has been proposed to be the underlying mechanism. However, cross-modal relationships especially between somatic and visceral pain have rarely been investigated even in healthy volunteers. Furthermore, psychological influences on pain have rarely been characterized across modalities. METHODS: Sixty-one healthy participants underwent testing of perception and pain thresholds for cutaneous thermode heat and cold, as well as for rectal balloon distension. Psychological testing for anxiety, depression, and pain experience (including catastrophizing and coping) as well as cardiac interoception was performed. Measurement quality and the correlations between the different modalities were examined. KEY RESULTS: Significant correlations existed between the perception thresholds for cold/heat (τB  = -0.28, p = 0.002) and cold/distension (τB  = -0.21, p = 0.03) and for the pain thresholds for cold/heat (r = -0.61, p < 0.001) and heat/distension (r = 0.33, p = 0.01). No association was found between pain thresholds and anxiety, depression, psychological experience with and processing of pain, or cardiac interoception. Retest reliabilities for pain measurements were satisfying after short intertrial intervals (all intraclass correlation coefficients >0.8), but less so after longer intervals. The individuals contributing to the respective correlations differ between measurements. CONCLUSIONS & INFERENCES: Moderate associations were found for pain thresholds across modalities. The strength of the associations and their stability over time warrants further investigation and might serve to increase the understanding of conditions affecting multiple pain modalities.

Intramolecular recognition in a jet-cooled short peptide chain: gamma-turn helicity probed by a neighbouring residue.

gamma-Turn, the shortest secondary structure of peptides, exists as two helical forms gamma(l) and gamma(d) of opposite handedness. The present gas phase study of capped l-Phe-Xxx peptides (Xxx = l-Ala, d-Ala or Aib: aminoisobutyric acid) provides a unique example of intramolecular chiral recognition of the gamma-turn helicity on Ala or Aib by the neighbouring residue Phe within the chain. With the chiral l- or d-Ala residues, the presence of a side-chain operates a discrimination between the two helical forms: one of them is widely favoured over the other (gamma(l) or gamma(d), respectively). This enables us to validate and calibrate the recognition capabilities of the nearby l-Phe residue. The discriminating interactions have been precisely characterized from their spectroscopic UV and IR signatures and identified by comparison with quantum chemistry calculations. Then, in the case of the non-chiral residue Aib, the two helical forms of the gamma-turn, which are simultaneously observed in the jet, have been discriminated and assigned by comparison with the chiral residues. The relative abundances of the diastereomeric forms l-Phe-Aib(gamma(l)) and l-Phe-Aib(gamma(d)) enable us to determine the most efficient recognition configuration.

Cloning and nucleotide sequence of the gene encoding the Ecal DNA methyltransferase.

The gene coding for the GGTNACC specific Ecal DNA methyltransferase (M.Ecal) has been cloned in E. coli from Enterobacter cloacae and its nucleotide sequence has been determined. The ecalM gene codes for a protein of 452 amino acids (Mr: 51,111). It was determined that M.Ecal is an adenine methyltransferase. M.Ecal shows limited amino acid sequence similarity to other adenine methyltransferases. A clone that expresses Ecal methyltransferase at high level was constructed.

Influence of different media and bloods on results of diffusion antibiotic susceptibility tests.

As part of an International Collaborative Study, the influence of medium and certain medium components on the results of disc susceptibility testing with nine antibiotics was examined and statistically evaluated. Four basic media, Trypticase Soy Agar, Grove and Randall's formula # 9 agar, and Mueller-Hinton agar with and without 1 mg of l-tryptophan per 100 ml, were used with six bacterial strains. Significant differences in zone diameters occurred with most antibiotics. The largest consistent differences were seen with tetracycline, and appeared to result from varying degrees of chelation of the antibiotic with free cations in the media. Reproducibility studies on different batches of Mueller-Hinton agar from two manufacturers showed some statistically significant differences, which were small except with tetracycline. Reproducibility between the products of a single manufacturer was excellent. The results of experiments to determine the effect of the addition of 5% blood of different species to agar medium showed that medium containing citrated or defibrinated horse, rabbit, sheep, or human blood yielded closely similar zone diameters, except that tetracycline zones were larger with citrated blood. A single type of medium should be selected for routine susceptibility tests whenever possible, and reproducibility of performance of the products of different manufacturers should be sought.