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Streptococcal plasmid pIP501 has a functional oriT site.

DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.

Base Sequence↗

Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1.

The DNA sequence for a 4.4-kb HindIII-XhoI Methylobacterium extorquens AM1 DNA fragment that is known to contain three genes (mxaAKL) involved in incorporation of calcium into methanol dehydrogenase (I. W. Richardson and C. Anthony, Biochem. J. 287:709-7115, 1992) was determined. Five complete open reading frames and two partial open reading frames were found, suggesting that this region contains previously unidentified genes. A combination of sequence analysis, mutant complementation data, and gene expression studies showed that these genes correspond to mxaSACKLDorf1. Of the three previously unidentified genes (mxaC, mxaD, and orf1), mutant complementation studies showed that mxaC is required for methanol oxidation, while the function of the other two genes is still unknown.

Alcohol Oxidoreductases↗

Newer antiepileptic drugs in bipolar disorder: rationale for use and role in therapy.

Antiepileptic drugs (AEDS) are used regularly in the treatment of patients with bipolar disorders. Carbamazepine and valproic acid (sodium valproate) are effective as antimanic treatments, and the success of these medications has prompted investigation of other AEDs as possible treatments in patients with mood disorders. Lamotrigine appears to be the most promising of the newer AEDs with respect to effects in mood disorders. Current evidence suggests efficacy of this drug both as monotherapy and as an adjunctive agent in bipolar depression, and studies are underway to clarify its efficacy in mood stabilisation and rapid cycling, as currently available data are equivocal. Use of gabapentin is not as well supported in the literature, although data from open trials using it as an adjunctive agent suggest that it may be helpful in patients with bipolar depression. There have been some open trials and case reports supporting the use of topiramate as an adjunctive agent for the treatment of mania; however, data from controlled trials are not yet available. Further controlled trials of lamotrigine, gabapentin or topiramate as monotherapy and adjunctive treatment are needed to clarify their potential roles in the treatment of patients with mood disorders.

Acetates↗

Molecular cloning and identification of a novel oxygenase gene specifically induced during the growth of Rhodococcus sp. strain T104 on limonene.

Rhodococcus sp. strain T104 is able to utilize both limonene and biphenyl as growth substrates. Furthermore, T104 possesses separate pathways for the degradation of limonene and biphenyl. Previously, we found that a gene(s) involved in limonene degradation was also related to indigo-producing ability. To further corroborate this observation, we have cloned and sequenced a 8,842-bp genomic DNA region with four open reading frames, including one for indole oxygenase, which converts indole to indigo (a blue pigment), The reverse transcription PCR data demonstrated that the identified indole oxygenase gene is specifically induced by limonene, thereby implicating this gene in the degradation of limonene by T104.

Cloning, Molecular↗

The test-retest reliability of a standardized neurocognitive and neurophysiological test battery: "neuromarker".

NeuroMarker combines EEG and ERP measures with neurocognitive tests in a fully computerized and standardized testing system. It is designed for use across the lifespan and has a large normative database of over 1,000 subjects. This study was a preliminary evaluation of "NeuroMarker" in subjects spanning four decades. Twenty-one healthy subjects (12-57 years) were tested at baseline and four weeks later. From the "Neuromarker" battery, the authors analyzed EEG data (eyes open and closed) and ERPs elicited during auditory oddball (N100, P200, N200, P300) and working memory (P150, P300) tasks. Concomitant neuropsychological data, acquired using a touch-screen system, comprised measures of sensori-motor, attention, verbal, executive, and memory function. Test-retest data were examined using analyses of variance and correlational procedures (corrected for multiple comparisons), with parallel analyses of age. EEG data did not differ across sessions, and showed high test-retest reliability (.71-.95), particularly for theta and delta (>.85). ERP components also showed sound reliability, particularly for sites where components are maximal: fronto-central N100 (.76-.77), centro-parietal P300 (.78-.81) to oddball targets, N100 and P200 (.74-.86) to oddball non-targets, and P150 amplitude and latency (.84-.93) to working memory stimuli. Neuropsychological tests showed a similarly sound level of consistency (on average, .70), with the most consistent tests tapping simple motor function, estimated intelligence, switching of attention (Part 2), verbal interference response time and memory intrusions (.71-.89). Age and sex did not have a differential impact on reliability for EEG, ERP, or neuropsychology measures. These findings provide preliminary evidence that the "NeuroMarker" battery is reliable for test-retest assessments. The results suggest that the standardized approach has utility for providing sensitive clinical and treatment evaluations across age groups.

Adolescent↗

Selection of virulence-associated determinants of Streptococcus suis serotype 2 by in vivo complementation.

Within Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. We introduced a genomic library of a pathogenic strain into a weakly pathogenic strain. After infection of the library into young piglets pathogenic transformants were selected. One specific transformant containing a 3-kb fragment of the pathogenic strain appeared to be dominantly enriched in diseased pigs. The observed enrichment was not tissue specific. The selected fragment, when introduced into two different weakly pathogenic strains, increased the virulence of these strains considerably. In contrast, introduction of the corresponding fragment of a weakly pathogenic strain had only minor effects on virulence. Nucleotide sequence analysis of the selected fragment of the pathogenic strain revealed the presence of two potential open reading frames, both of which were found to be mutated in the corresponding fragment of the weakly pathogenic strain. These data strongly suggest that the selected fragment contains determinants important for virulence.

Amino Acid Sequence↗

Molecular cloning and characterization of gravity specific cDNA in rice (Oryza sativa L.) suspension callus.

Rice (Oryza sativa L. var. Nipponbare) suspension callus was exposed to gravity stress at 450,000 g for 2 hours, after which poly(A)+RNA was isolated and a cDNA library was constructed. Three different gravity specific cDNAs, namely, GSC 128, GSC 233 and GSC 381 of 0.67, 0.60 and 0.68 kilobase pairs and transcripts of 1.9, 1.6 and 2.0 kb, respectively, were isolated by differential screening and Northern hybridization. The maximum level of transcript was achieved after 4 hours of exposure to gravity at 450,000 g for GSC 128, 2 hours for GSC 233 and 8 hours for GSC 381 followed by a gradual decrease to undetectable levels with the extension of gravitation time. Callus (GSC 128), shoot and callus (GSC 381) and root and callus (GSC 233) specific expression of transcripts was identified. Although the protection of callus by treatment with ABA, kinetin and sucrose extended the period of expression of mRNA in suspension callus after gravity exposure, the expression of gravity-inducible mRNA was exclusively regulated by the degree of callus viability or survival after the stress. In addition, we demonstrated that the level of GSC 381 transcript was markedly increased by exposing the cell to periodical gravity stress, suggesting that this mRNA is expressed and translated into special proteins which are closely related to the survival of the cell against gravity stress. The sequence of GSC 233 and GSC 381, consisting of 417 and 531 base pairs of the longest open reading frames, encode polypeptides with calculated molecular weights of 15.29 and 19.47 kDa, respectively. A sequence homology search against a data bank revealed that GSC 233 and GSC 381 differed from other stress inducible genes in terms of the coding sequence and expression characteristics.

Adaptation, Physiological↗

Topiramate in the long-term treatment of binge-eating disorder associated with obesity.

BACKGROUND: This study assessed the long-term effectiveness and tolerability of topiramate in binge-eating disorder (BED) with obesity. METHOD: Sixty-one patients with BED (DSM-IV-TR criteria) and obesity enrolled in a 14-week, single-center, randomized, double-blind, placebo-controlled study. Completers (N = 35) were offered participation in a 42-week, open-label extension trial of topiramate. Fifteen patients who received topiramate and 16 patients who received placebo in the double-blind study entered the open-label trial. Topiramate was titrated from 25 mg/day to a maximum of 600 mg/day. The primary endpoint was change from baseline to final visit in weekly binge frequency using the last observation carried forward for all patients who received topiramate. Baseline for patients receiving double-blind topiramate was the beginning of the controlled study; for patients receiving placebo, baseline was the beginning of the open-label trial. Open-label data were gathered from December 1998 to November 2000. RESULTS: Forty-four patients (31 who received topiramate in the open-label trial plus 13 who received topiramate in the double-blind study only) received at least 1 dose of topiramate; 43 patients provided outcome measures at a median final dose of 250 mg/day. Mean weekly binge frequency declined significantly from baseline to final visit for all 43 patients (-3.2; p < .001), for the 15 patients who received topiramate during the controlled and open-label studies (-4.0; p < .001), and for the 15 patients who received topiramate only during the open-label trial (-2.5; p = .044). Patients also exhibited statistically significant reduction in body weight. The most common reasons for topiramate discontinuation were protocol nonadherence (N = 17) and adverse events (N = 14). CONCLUSION: Topiramate treatment was associated with enduring improvement in some patients with BED and obesity but was also associated with a high discontinuation rate.

Adolescent↗

Standard interchange for computerized electrocardiography.

In the SCP-ECG project (AIM #1015) of the pilot AIM Programme a standard communications protocol was developed for computerized electrocardiography, consisting of standards for the interchange, encoding and storage of digital ECG data. Part of this work is continued under the main AIM Programme (January 1992- December 1994) as AIM Project #2026, entitled "Open European Data Interchange and Processing for Computerized Electrocardiography (OEDIPE)" and was taken up by the CEN/TC 251 Committee as a priority item, for implementation into an official European Standard in Health Care Informatics. The objectives and implementation of this Standard are described in the present paper.

Electrocardiography↗

The DNA sequence of cosmid 14-13b from chromosome XIV of Saccharomyces cerevisiae reveals an unusually high number of overlapping open reading frames.

This work is part of the effort for sequencing chromosome XIV of Saccharomyces cerevisiae. Cosmid 14-13b contains a 37.8 kb insert derived from a partial Sau3A digestion of the genome, cloned into the BamHI site of the vector Pou6. The strategy used for sequencing is based on the fragmentation of the whole cosmid by sonication, followed by shotgun sequencing on an Applied Biosystem DNA sequencer. The clones with inserts corresponding to the vector were identified by dot-blot hybridization, without the need of sequencing. The analysis of the DNA sequence reveals 29 open reading frames (ORFs) longer than 300 bases. Nine ORFs are internal to some other ORFs. Similarity searches against DNA and protein data banks show that six ORFs correspond to already known yeast genes (OMP1, PSU1, MLS1, RPC19, DBP2, CYB5) and one ORF matches the sequence of a putative yeast gene (ESBP6).

Chromosome Mapping↗

Analysis of a 70 kb region on the right arm of yeast chromosome II.

In the framework of the EC programme for sequencing yeast chromosome II, we have determined the nucleotide sequence of a 70 kb region. Subsequent analysis revealed 35 open reading frames, 14 of which correspond to known yeast genes. From structural parameters and/or similarity searches with entries in the current data libraries, a preliminary functional assessment of several of the putative novel gene products can be made. The gene density in this region amounts to one gene in 1.98 kb. Coding regions occupy 75% of the total DNA sequence. Within the intergenic regions, potential regulatory elements can be predicted. The data obtained here may serve as a basis for a more detailed biochemical analysis of the novel genes.

Amino Acid Sequence↗

Cloning of an adenosine A1 receptor-encoding gene from rabbit.

A partial cDNA encoding the A1 adenosine receptor (A1AR), which lacks nucleotides coding for the first 74 amino acids (aa), was isolated from a rabbit kidney cDNA library. The missing 5' end sequence was obtained from an overlapping rabbit genomic clone which was found to contain the flanking 5' untranslated region (5'UTR), the first exon and part of the first intron. Together, the cDNA and genomic clones provide the entire open reading frame (ORF) encoding rabbit A1AR. The deduced aa sequence is highly homologous to the canine, rat and bovine A1ARs. These data also indicate that the A1AR gene belongs to the family of intron-containing G-protein-linked receptor genes.

Adenosine↗

In vitro marginal fit of three all-ceramic crown systems.

STATEMENT OF PROBLEM: Studies on marginal discrepancies of single restorations using various systems and materials have resulted in statistical inferences that are ambiguous because of small sample sizes and limited numbers of measurements per specimen. PURPOSE: The purpose of this study was to compare the marginal adaptation of single anterior restorations made using different systems. MATERIAL AND METHODS: The in vitro marginal discrepancies of 3 different all-ceramic crown systems (Celay In-Ceram, conventional In-Ceram, and IPS Empress 2 layering technique), and a control group of metal ceramic restorations were evaluated and compared by measuring the gap dimension between the crowns and the prepared tooth at the marginal opening. The crowns were made for 1 extracted maxillary central incisor prepared with a 1-mm shoulder margin and 6-degree tapered walls by milling. Thirty crowns per system were fabricated. Crown measurements were recorded with an optical microscope, with an accuracy of +/-0.1 microm, at 50 points spaced approximately 400 microm along the circumferential margin. The criterion of 120 microm was used as the maximum clinically acceptable marginal gap. Mean gap dimensions and standard deviations were calculated for marginal opening. The data were analyzed with a 1-way analysis of variance (alpha=.05). RESULTS: Mean gap dimensions and standard deviations at the marginal opening for the incisor crowns were 87 +/- 34 microm for control, 83 +/- 33 microm for Celay In-Ceram, 112 +/- 55 microm for conventional In-Ceram, and 46 +/- 16 microm for the IPS Empress 2 layering technique. Significant differences were found among the crown groups (P<.05). Compared with the control group, the IPS Empress 2 group had significantly smaller marginal discrepancies (P<.05), and the conventional In-Ceram group exhibited significantly greater marginal discrepancies (P<.05). There was no significant difference between the Celay In-Ceram and the control group. CONCLUSION: Within the limitations of this study, the marginal discrepancies were all within the clinically acceptable standard set at 120 microm. However, the IPS Empress 2 system showed the smallest and most homogeneous gap dimension, whereas the conventional In-Ceram system presented the largest and more variable gap dimension compared with the metal ceramic (control) restoration.

Aluminum Oxide↗

Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes.

Purification of endosomal/lysosomal vesicles of Trypanosoma brucei brucei bloodstream forms and generation of monoclonal antibodies led to the isolation of antibodies directed against an 85 kDa, Golgi and endocytic traffic-associated protein termed tGLP-1, Trypanosoma Golgi/lysosome protein-1. Preliminary immunoelectron microscopical analysis revealed that the protein is present in, but not restricted to, the limiting membrane of multivesicular lysosomes and is more abundant in bloodstream forms compared to the procyclic stage. The corresponding gene was cloned and is present as a single copy. Blast searches did not reveal any homologies to other proteins and genes published. The nucleotide sequence of the gene (1848 base pairs) predicted a type 1 membrane topology with an N-terminal signal sequence (20 aa), a luminal domain with 2 N-glycosylation sites (524 aa), a transmembrane domain (23 aa), and a long cytosolic tail domain (49 aa). Polyclonal antibodies raised against the cytosolic tail confirmed the localization of the gene product to multivesicular lysosomes but revealed that the majority of the protein was in the Golgi apparatus. Colabelling with an antibody against p67, a lysosomal glycoprotein of trypanosomes, revealed extensive overlap between the proteins with opposing relative abundance. Expression of the tGLP-1 open reading frame in Leishmania resulted in Golgi localization, and in Toxoplasma, in localization to both the Golgi and endoplasmic reticulum. These data indicate conservation in the functionality of the Golgi-targeting sequence of tGLP-1.

Amino Acid Sequence↗

Random sequencing of Paramecium somatic DNA.

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.

Amino Acid Sequence↗

Sexuality after hysterectomy: a model based on quantitative and qualitative analysis of 104 women before and after subtotal hysterectomy.

To study the effect of subtotal hysterectomy on a woman's sexual life 104 women were interviewed 1 month before and 1 year after subtotal hysterectomy. The interview was semi-structured with the addition of unstructured, open questions. Data concerning sexuality before and after the operation were evaluated with statistical methods, and the result was compared to the answers to the open questions. The most important factor for postoperative sexuality was preoperative sexual activity and enjoyment. Most women described the meaning of sexuality in terms of partner relationship. Those who reported improved sexuality in most cases described the improvement in terms of improved personal sexual capacity, whereas most cases of deterioration described the deterioration in terms of absent, poor, or malfunctioning partner relationship. A model for sexuality after hysterectomy is introduced. Sexual functioning before the operation is the most important factor. Partner sexual functioning, socioemotional support from the partner, adaptation to the consequences of the hysterectomy, mental status, physical ability and hormonal status are other factors influencing sexuality after hysterectomy.

Adult↗

Complete nucleotide sequence and analysis of a novel human papillomavirus (HPV 84) genome cloned by an overlapping PCR method.

Molecular diagnosis of human papillomaviruses (HPVs) in cervicovaginal samples reveals a plethora of known and novel HPV genomes. We describe the use of an overlapping PCR method to clone and analyze the complete genome of HPV 84 from cervicovaginal cells obtained from a 21-year-old Caucasian female with a normal Pap smear. The 7948-bp complete nucleotide sequence of HPV 84 was determined from five overlapping PCR products by sequence walking. A BLAST homology search demonstrated that HPV 84 was most closely related to HPV 61 (89%), HPV 72 (86%), and HPV 83 (85%) by nucleotide sequence analysis of the L1 open reading frame, placing it in the HPV genome homology group A3. Previously, this virus had been identified as Pap155. Based on extensive epidemiological data, HPV 84 is a highly prevalent genital papillomavirus primarily detected in normal and HIV-infected women.

Adult↗

Multimerization of the ligand binding domains of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a beta roll followed by two alpha helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.

Animals↗