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PubMed1991

Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis.

A method which allowed the stable integration of DNA fragments at a single site (attB) in the chromosome of Mycobacterium smegmatis was developed using an integrative element from Streptomyces ambofaciens, pSAM2. Vectors containing an Escherichia coli replicon (pBR322), the kanamycin resistance gene from Tn903 for selection in mycobacteria, and a fragment of pSAM2 containing the int gene as well as the attachment site (attP) were constructed and introduced to M. smegmatis by electroporation. Transformants showed stable integration of the plasmid into a single site (attB) of the mycobacterial genome. This approach should be valuable for analyses of gene expression in various mycobacterial species and permit the development of stable recombinant mycobacterial vaccine strains expressing bacterial or viral genes inserted in pSAM2.

Blotting, Southern↗
PubMed2004

Abnormal global processing along the dorsal visual pathway in autism: a possible mechanism for weak visuospatial coherence?

Frith and Happe (Frith, U., & Happe, F. (1994). Autism: Beyond theory of mind. Cognition, 50, 115-132) argue that individuals with autism exhibit 'weak central coherence': an inability to integrate elements of information into coherent wholes. Some authors have speculated that a high-level impairment might be present in the dorsal visual pathway in autism, and furthermore, that this might account for weak central coherence, at least at the visuospatial level. We assessed the integrity of the dorsal visual pathway in children diagnosed with an autism spectrum disorder (ASD), and in typically developing children, using two visual tasks, one examining functioning at higher levels of the dorsal cortical stream (Global Dot Motion (GDM)), and the other assessing lower-level dorsal stream functioning (Flicker Contrast Sensitivity (FCS)). Central coherence was tested using the Children's Embedded Figures Test (CEFT). Relative to the typically developing children, the children with ASD had shorter CEFT latencies and higher GDM thresholds but equivalent FCS thresholds. Additionally, CEFT latencies were inversely related to GDM thresholds in the ASD group. These outcomes indicate that the elevated global motion thresholds in autism are the result of high-level impairments in dorsal cortical regions. Weak visuospatial coherence in autism may be in the form of abnormal cooperative mechanisms in extra-striate cortical areas, which might contribute to differential performance when processing stimuli as Gestalts, including both dynamic (i.e., global motion perception) and static (i.e., disembedding performance) stimuli.

Autistic Disorder↗
PubMed2006

A measuring system for the fast simultaneous isotope ratio and elemental analysis of carbon, hydrogen, nitrogen and sulfur in food commodities and other biological material.

The isotope ratio of each of the light elements preserves individual information on the origin and history of organic natural compounds. Therefore, a multi-element isotope ratio analysis is the most efficient means for the origin and authenticity assignment of food, and also for the solution of various problems in ecology, archaeology and criminology. Due to the extraordinary relative abundances of the elements hydrogen, carbon, nitrogen and sulfur in some biological material and to the need for individual sample preparations for H and S, their isotope ratio determination currently requires at least three independent procedures and approximately 1 h of work. We present here a system for the integrated elemental and isotope ratio analysis of all four elements in one sample within 20 min. The system consists of an elemental analyser coupled to an isotope ratio mass spectrometer with an inlet system for four reference gases (N(2), CO(2), H(2) and SO(2)). The combustion gases are separated by reversible adsorption and determined by a thermoconductivity detector; H(2)O is reduced to H(2). The analyser is able to combust samples with up to 100 mg of organic material, sufficient to analyse samples with even unusual elemental ratios, in one run. A comparison of the isotope ratios of samples of water, fruit juices, cheese and ethanol from wine, analysed by the four-element analyser and by classical methods and systems, respectively, yielded excellent agreements. The sensitivity of the device for the isotope ratio measurement of C and N corresponds to that of other systems. It is less by a factor of four for H and by a factor of two for S, and the error ranges are identical to those of other systems.

Carbon↗
PubMed1995

Does thermoregulatory feeding occur in newborn infants? A novel view of the role of brown adipose tissue thermogenesis in control of food intake.

The physiological significance of the extensive deposits of brown adipose tissue (BAT) in newborn human infants has been the subject of much experimentation and discussion. Because of its large thermogenic capacity, its function has usually been viewed as preparing the infant for producing heat in response to cold exposure at birth. Newborn infants are indeed capable of precise thermoregulation for a limited time over a rather limited range of ambient temperatures, from thermoneutrality (32-34 degrees C) down to common "room" temperatures (24-28 degrees C). During such mild "cold-exposure", in response to a decrease in their skin temperature, their sympathetic nervous system activity increases, and they can more than double their resting metabolic rate, principally by thermogenesis in their BAT. This review puts forward an entirely new role for BAT thermogenesis in the cyclic feeding pattern of newborn infants during their first months of life. BAT thermogenesis is proposed to be an integral element in a physiological thermoregulatory feeding control mechanism in which extended periods of very gradual cooling are interspersed with episodes of increased sympathetic nervous system activity, increased heating via BAT thermogenesis, arousal, and feeding. The cry with which the baby attracts its mother's attention is an integral part of the mechanism, as is the nutritive suckling reflex and the behavior of the mother. Initiation of feeding is attributed to a transient dip in blood glucose concentration that is due to stimulation of glucose utilization in the BAT. Termination of feeding is attributed to the high temperature brought about by the stimulated BAT thermogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue, Brown↗
PubMed1991

Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects.

A large number of insect species have been screened for the presence of the retrotransposable elements R1 and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to greater than 50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to R1 or R2 elements. Surprisingly, many species appeared to contain highly divergent copies of R1 and R2 elements. For example, a parasitic wasp contained at least four families of R1 elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.

Amino Acid Sequence↗
PubMedDate not supplied

Oral diseases and conditions throughout the lifespan. I. Diseases and conditions directly associated with tooth loss.

This is the first of two articles providing an overall view of oral diseases and conditions, taking the oral cavity and its organs and tissues as integrated parts of the human body. This article provides an overview of three oral diseases and conditions that are associated directly with loss of tooth structure or loss of teeth and reviews their risk factors and preventive and treatment interventions. Two of these conditions, dental caries and periodontal diseases (or their sequelae), remain the main cause of tooth loss in the U.S. Prevention, treatment, and control of these diseases require many hours of training during dental education and many hours of treatment in dental practice. The profession has fragmented into specialties based on diseases; as a result, many dentists fail to see them as integral elements of the human experience. As primary care specialists, dentists must be reminded that they need to make prevention the first choice, provide the best treatment possible based on available scientific evidence, and keep informed of new advances in research while always remembering that the oral cavity is an integral part of the human body. The changes and trends observed in the epidemiology of oral diseases will demand new skills in our dental graduates. Future dental graduates may achieve a solid understanding of oral diseases at both the biological and population level by using oral tissues and saliva to diagnose systemic diseases, relying on medical facilities to order laboratory tests, and diagnosing and treating patients in close collaboration with their medical colleagues.

Dental Caries↗
PubMed1998

Transfer of Tn5385, a composite, multiresistance chromosomal element from Enterococcus faecalis.

Tn5385 is a ca. 65-kb element integrated into the chromosomes of clinical Enterococcus faecalis strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via beta-lactamase production. Tn5385 is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn5385 have been identified, including conjugative transposon Tn5381, composite transposon Tn5384, and elements indistinguishable from staphylococcal transposons Tn4001 and Tn552. The divergent regions of Tn5385 are linked by a series of insertion sequence (IS) elements (IS256, IS257, and IS1216) of staphylococcal and enterococcal origin. The ends of Tn5385 consist of directly repeated copies of enterococcal IS1216. Within the chromosomes of strains CH19 and CH116, Tn5385 has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn5385 from both donors into E. faecalis recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn5385 in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn5385 insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn5385 from the CH19 chromosome by recombination across the terminal IS1216 elements and insertion into the recipient chromosome by recombination across Tn5381 (within Tn5385) and a previously transferred Tn5381 copy in the recipient chromosome. These data confirm that Tn5385 is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.

Base Sequence↗
PubMed2000

CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes.

CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae. CTX prophages in O1 El Tor and O139 strains of V. cholerae are found within arrays of genetically related elements integrated at a single locus within the V. cholerae large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi. In contrast, O1 classical strains of V. cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V. cholerae. Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of V. cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.

Bacteriophages↗
PubMed1986

An RNA polymerase I enhancer in Saccharomyces cerevisiae.

By the use of an artificial gene coding for rRNA (rDNA gene), we found that transcription of the major precursor rRNA in Saccharomyces cerevisiae cells is stimulated 15-fold by a positive control element located 2 kilobases upstream of the transcription initiation site. Analysis of in vitro runon transcripts suggests that this promoter element increases the frequency of initiation by RNA polymerase I molecules. A 190-base-pair fragment encompassing the promoter element can stimulate transcription on a centromere plasmid in either orientation, upstream or downstream of the transcription initiation site, suggesting that it is an enhancer element. Integration of artificial rDNA genes into a nonribosomal locus in the genome demonstrates that the rDNA enhancer functions either 5' or 3' to an rRNA transcription unit, suggesting it may operate in both directions within the rDNA tandem array. This is the first observation in S. cerevisiae of the stimulation of transcription by an element placed downstream. Finally, enhancer activity is dependent upon sequences that lie at both boundaries of the 190-base-pair fragment. In particular, a 5-base-pair deletion at the extreme 3' boundary of the 190-base-pair fragment greatly reduces the activation of transcription and implicates a set of inverted repeats.

Base Sequence↗
PubMed2005

Tn5386, a novel Tn916-like mobile element in Enterococcus faecium D344R that interacts with Tn916 to yield a large genomic deletion.

We describe Tn5386, a novel ca.-29-kb Tn916-like mobile element discovered to occur in ampicillin-resistant, Tn916-containing Enterococcus faecium D344R. PCR amplification experiments after overnight growth with or without tetracycline revealed "joint" regions of circularized Tn5386 composed of 6-bp sequences linking different transposon termini. In one case (no tetracycline), the termini were consistent with those derived by target site analysis of the integrated element. In the other case, the termini were virtually identical in distance from the integrase binding regions, as seen with Tn916. These data are consistent with a model in which one PCR product results from the action of Tn5386 integrase, whereas the other results from the action of the Tn916 integrase on Tn5386. Spontaneous conversion of D344R to an ampicillin-susceptible phenotype (D344SRF) was associated with a 178-kb deletion extending from the left end of Tn5386 to the left end of Tn916. Examination of the Tn5386 junction after the large deletion event suggests that the deletion resulted from an interaction between the nonintegrase ends of Tn5386 and Tn916. The terminus of Tn5386 identified in this reaction suggested that it may have resulted from the activity of the Tn916 integrase (Int(Tn916)). The "joint" of the circular element resulting from this excision was amplifiable from D344R, the sequence of which revealed a heteroduplex consistent with Int(Tn916)-mediated excision. In contrast, Tn5386 joints amplified from ampicillin-susceptible D344SRF revealed ends consistent with Tn5386 integrase activity, reflecting the absence of Tn916 from this strain. Tn5386 represents a new member of the Tn916 transposon family. Our data suggest that excision of Tn5386 can be catalyzed by the Tn916 integrase and that large genomic deletions may result from the interaction between these heterologous elements.

Ampicillin Resistance↗
PubMed2001

[Human cloning from the perspective of The Council of Europe bioethical standards].

Allegations negating the role of law in the resolving the controversial problem of human cloning are unjustified. Human rights, implying the magnitude and value of human person and deeply rooted inherent dignity of the human being, constitute the very foundation of every legal order. Every lawyer, as well as every specialist in medicine or biology must be aware of his own human dignity and the ethical consequences. Among the standards of the Council of Europe in the context of human cloning there must be mentioned the Additional Protocol of January 12, 1998 on the Prohibition of Cloning Human Beings. It is the integral element of the normative system of the mother convention, the European Bioethical Convention of April 4, 1997. It has the distinguished place - within this system as far as its substantial provisions exclude the possibilities of limitations and derogation. The system of the Convention and the Protocol must be viewed in the light of a broader normative environment, including the integral system of the European Convention on Human Rights and the set of recommended bioethical standards. The absolute prohibition embodied in the Protocol is limited to all the methods leading to the creation of genetically identical human beings. The protocol does not directly regulate cloning of human tissues and cells, including embryonic stem cells. However, some conclusions may be taken from the Convention and from the recommended standards. It is the assumption of the Convention and the Protocol that the guarantees embodied there must be apprehended as practical and effective ones, justifiable and not excluding the use of proper sanctions by the State. It is an unacceptable view that scientists are excluded from the sphere of the functioning of the above-mentioned prohibition. The real sense of this prohibition is to stop the unlimited liberty and arbitrary practice of such scientists. The freedom of scientific research is not absolute, and must be guided by the respect for human dignity and human rights, as by well as protective guarantees embodied in the Convention and recommended standards.

Bioethics↗
PubMed2002

The role of Bacteroides conjugative transposons in the dissemination of antibiotic resistance genes.

Investigations into the mechanisms of antibiotic resistance gene transfer utilized by Bacteroides species have led to a greater understanding of how bacteria transfer antibiotic resistance genes, and what environmental stimuli promote such horizontal transfer events. Although Bacteroides spp. harbor a variety of transmissible elements that are involved in the dissemination of antibiotic resistance genes, it is one particular class of elements, the conjugative transposons, that are responsible for most of the resistance gene transfer in Bacteroides. The potential for Bacteroides conjugative transposons to transfer antibiotic resistance genes extends beyond those genes carried by the conjugative transposon itself, because Bacteroides conjugative transposons are able to mobilize coresident plasmids in trans and in cis, and also stimulate the excision and transfer of unlinked integrated elements called mobilizable transposons. These characteristics of conjugative transposons alone have significant implications for the ecology and spread of antibiotic resistance genes, and in terms of biotechnology. A novel feature of the most widespread family of Bacteroides conjugative transposons, the CTnDOT/ERL family, is that their transfer is stimulated 100- to 1000-fold by low concentrations of tetracycline. This is significant because the use of antibiotics not only selects for resistant Bacteroides strains, but also stimulates their transfer. Other Bacteroides conjugative transposons do not require any induction to stimulate transfer, and hence appear to transfer constitutively. The constitutively transferring elements characterized so far appear to have a broader host range than the CTnDOT/ERL family of conjugative transposons, and the prevalence of these elements is on the increase. Since these constitutively transferring elements do not require induction by antibiotics to stimulate transfer, they have the potential to become as pervasive as the CTnDOT/ERL family of conjugative transposons.

Anti-Bacterial Agents↗
PubMed2003

Transferable vanB2 Tn5382-containing elements in fecal streptococcal strains from veal calves.

Three vancomycin-resistant veal calf fecal streptococci, identified as Streptococcus gallolyticus (n = 2) and Streptococcus lutetiensis, were shown to harbor vanB2 Tn5382-like elements earlier described in enterococci. One S. gallolyticus strain had a 1,495-bp IS256-related element inserted in vanS(B). The vanB2 Tn5382 element present in the plasmid-free S. lutetiensis strain was transferable to Enterococcus faecium BM4105-RF, Enterococcus faecalis JH2-2, and its recombination-deficient derivative, UV202. The transfer frequencies were comparable between recipient strains (from 1 x 10(-7) to 7 x 10(-6)). All transconjugants acquired a vanB-containing chromosomal insert of approximately 100 kb, apparently by site-specific integration. Secondary transconjugants were not observed in intraspecies retransfer experiments. These observations are consistent with a conjugative, selftransmissible, integrative element that might be involved in the interspecies spread of vanB2 resistance determinants. Two JH2-2-derived transconjugants had also gained additional copies of large vanB-containing chromosomal fragments, a process that involves unexplained mechanisms that seems to require functional host cell-dependent recombination mechanisms.

Animals↗
PubMed2004

Epigenetic control of CACTA transposon mobility in Arabidopsis thaliana.

Epigenetic mutation, heritable developmental variation not based on a change in nucleotide sequence, is widely reported in plants. However, the developmental and evolutionary significance of such mutations remains enigmatic. On the basis of our studies of the endogenous Arabidopsis transposon CACTA, we propose that the inheritance of epigenetic gene silencing over generations can function as a transgenerational genome defense mechanism against deleterious movement of transposons. We previously reported that silent CACTA1 is mobilized by the DNA hypomethylation mutation ddm1 (decrease in DNA methylation). In this study, we report that CACTA activated by the ddm1 mutation remains mobile in the presence of the wild-type DDM1 gene, suggesting that de novo silencing is not efficient for the defense of the genome against CACTA movement. The defense depends on maintenance of transposon silencing over generations. In addition, we show that the activated CACTA1 element transposes throughout the genome in DDM1 plants, as reported previously for ddm1 backgrounds. Furthermore, the CACTA1 element integrated into both the ddm1-derived and the DDM1-derived chromosomal regions in the DDM1 wild-type plants, demonstrating that this class of transposons does not exhibit targeted integration into heterochromatin, despite its accumulation in the pericentromeric regions in natural populations. The possible contribution of natural selection as a mechanism for the accumulation of transposons and evolution of heterochromatin is discussed.

Arabidopsis↗
PubMed2005

Numerical simulation of scanning electrochemical microscopy experiments with frame-shaped integrated atomic force microscopy--SECM probes using the boundary element method.

Integrated submicroelectrodes for combined AFM-SECM measurements are characterized with numerical simulations using the boundary element method. SECM approach curves and SECM images are calculated and analyzed for a model substrate containing pronounced topographical and electrochemical features. The theoretically calculated image has been compared to the experimental data and shows excellent quantitative agreement. Hence, the applicability of integrated AFM-SECM electrodes for combined electrochemical and topographical imaging and a profound theoretical description including quantification of the obtained results are demonstrated.

Electrochemistry↗
PubMed2006

Evaluation of clonality in enterococci isolated in Brazil carrying Tn1546-like elements associated with vanA plasmids.

Fifty-one vancomycin-resistant enterococci samples isolated from different geographic regions in Brazil were studied. All the isolates harboured the vanA gene as demonstrated by PCR analysis, and in a majority of strains the gene was associated with a transferable plasmid of 70 kb. A single variant of the prototype Tn1546 associated with common transferable vanA-containing plasmids has spread among the enterococcal strains circulating in Brazil. The VanA element integrity in these enterococci strains and the different pulsed-field gel electrophoresis patterns suggest horizontal transmission of the vancomycin resistance transposon in Brazilian strains.

Bacterial Proteins↗
PubMed2003

The CIP (comprehensive integrative puzzle) assessment method.

This paper describes a novel tool for assessment in medical education, the comprehensive integrative puzzle (CIP). The dual scoring system of the puzzle stresses the integrative elements of diagnostic thinking and clinical reasoning, while preserving the ability to discern proficiency in various disciplinary elements. The CIP has the format of an 'extended matching' crossword puzzle. Its answer sheet is a grid comprising rows and columns. The left-hand column contains diagnoses or brief clinical vignettes. To complete the cells of the grid the student is required to match, stepwise, the various 'disciplinary investigations' to the diagnoses or clinical vignettes. When the puzzle is completed each horizontal row reflects a coherent medical case. The completed horizontal rows reflect integrative ability (diagnostic thinking and clinical reasoning) and the vertical columns measure the student's proficiency in interpreting medical history data, physical examination findings, laboratory test results, ECG, imaging, special tests, pathology and pharmacology. The CIP has been well accepted by teachers and students during the last seven years at the Bruce Rappaport Faculty of Medicine in Haifa, and it has favorably affected both student assessment and teaching. The reliability of the test and its validity will be reported separately.

Cardiology↗
PubMed2002

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.

Bacteriophages↗