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PubMed2000

Functional corticospinal projections are established prenatally in the human foetus permitting involvement in the development of spinal motor centres.

From studies of subhuman primates it has been assumed that functional corticospinal innervation occurs post-natally in man. We report a post-mortem morphological study of human spinal cord, and neurophysiological and behavioural studies in preterm and term neonates and infants. From morphological studies it was demonstrated that corticospinal axons reach the lower cervical spinal cord by 24 weeks post-conceptional age (PCA) at the latest. Following a waiting period of up to a few weeks, it appears they progressively innervate the grey matter such that there is extensive innervation of spinal neurons, including motor neurons, prior to birth. Functional monosynaptic corticomotoneuronal projections were demonstrated neurophysiologically from term, but are also likely to be present from as early as 26 weeks PCA. At term, direct corticospinal projections to Group Ia inhibitory interneurons were also confirmed. Independent finger movements developed much later, between 6 and 12 months post-natally. These data do not support the proposal that in man, establishment of functional corticomotoneuronal projections occurs immediately prior to and provides the capacity for the expression of fine finger movement control. We propose instead that such early corticospinal innervation occurs to permit cortical involvement in activity dependent maturation of spinal motor centres during a critical period of perinatal development. Spastic cerebral palsy from perinatal damage to the corticospinal pathway secondarily involves disrupted development of spinal motor centres. Corticospinal axons retain a high degree of plasticity during axon growth and synaptic development. The possibility therefore exists to promote regeneration of disrupted corticospinal projections during the perinatal period with the double benefit of restoring corticospinal connectivity and normal development of spinal motor centres.

Autopsy↗

PubMed1997

Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.

Hepatocyte growth factor induces proliferation, motility and differentiation of epithelial cells through the tyrosine kinase receptor encoded by the MET protooncogene. The cytoplasmic portion of Met (referred to as cyto-Met) is activated but only weakly transforming. In order to determine the effect of activated Met on hepatocytes, we have targeted truncated Met expression to the liver by incorporating the cDNA into a vector carrying the entire human alpha-1-antitrypsin transcriptional unit. Transgenic expression in the liver of truncated human Met, containing the regulatory and the catalytic cytoplasmic domains, renders hepatocytes constitutively resistant to apoptosis and reproducibly permits immortalization. The emerging stable cell lines are not transformed and maintain a highly differentiated phenotype judged by the retention of epithelial cell polarity and the expression of hepatocyte-enriched transcription factors as well as hepatic products.

Animals↗

PubMed1979

A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae.

Studies of heterothallic and homothallic strains of Saccharomyces cerevisiae have led to the suggestion that mating-type information is located at three distinct sites on chromosome 3, although only information at the mating-type (MAT) locus is expressed (Hicks, Strathern and Herskowitz, 1977). We have found that the recessive mutation cmt permits expression of the normally silent copies of mating-type information at the HMa and HM alpha loci. In haploid strains carrying HMa and HM alpha, the cmt mutation allows the simultaneous expression of both a and alpha information, leading to a nonmating ("MATa/MAT alpha") phenotype. The effects of cmt can be masked by changing the mating-type information at HMa or HM alpha. For example, a cell of genotype MATa hma HM alpha cmt has an a mating type, while a MAT alpha hma HM alpha cmt strain is nonmating. Expression of mating-type information at the HM loci can correct the mating and sporulation defects of the mata* and mat alpha 10 alleles. Meiotic segregants recovered from cmt/cmt diploids carrying the mat mutations demonstrate that these mutants are not "healed" to normal MAT alleles, as is the case in parallel studies using the homothallism gene HO.--All of the results are consistent with the notion that the HMa and hm alpha alleles both code for alpha information, while HM alpha and hma both code for a information. The cmt mutation demonstrates that these normally silent copies of mating-type and sporulation information can be expressed and that the information at these loci is functionally equivalent to that found at MAT. The cmt mutation does not cause interconversions of mating-type alleles at MAT, and it is not genetically linked to MAT, HMa, HM alpha or HO. In cmt heterozygotes, cmt becomes homozygous at a frequency greater than 1% when the genotype at the MAT locus is mata*/MAT alpha or mat alpha 10/MATa.

Alleles↗

PubMed2006

Human fetal ovarian culture permits meiotic progression and chromosome pairing process.

BACKGROUND: The female meiotic process seems to be crucial for aneuploidy in humans. The first stages of mammalian female meiosis take place during the fetal period. Therefore, only little is known about female meiosis. The goal of this study was to develop a culture technique that permits human oocytes to progress through meiotic prophase, to provide a system to study human female meiosis. METHOD: Fetal ovaries from four cases were cultured up to 35 days in alpha-minimal essential medium, 2% human serum albumin, 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium and 100 IU/ml penicillin-100 microg/ml streptomycin. RESULTS AND CONCLUSIONS: Although ovarian response to culture conditions varied, human oocytes survived in vitro up to 5 weeks. In three cases, we observed significant variation in stages of meiosis among the cultures. The homologous chromosome pairing process was studied for the first time in cultured oocytes, and the results suggested that the pairing process was completed following the same features described previously for euploid oocytes, as followed by the chromosome-13 pairing process and synaptonemal complex formation. Although a higher proportion of degenerated oocytes were observed as culture time increased, we also observed oogonial entrance to meiotic prophase.

Cell Cycle↗

PubMed1993

Enhanced positive selection of a transgenic TCR by a restriction element that does not permit negative selection.

Very little is known about the conformational properties of the MHC molecules that are able to signal positive selection of a given TCR. To try to understand these parameters and to determine whether these requirements are shared with interactions during negative selection and antigen recognition, we have studied selection and antigen recognition of a transgenic TCR (specific for lymphocytic choriomeningitis virus glycoprotein and H-2Db) in the context of two Db mutants, H-2bm13 and H-2bm14. The data showed that the transgenic TCR was not positively selected by the H-2bm14 haplotype but, interestingly, enhanced positive selection was seen in H-2bm13 mice. The transgenic TCR could not be negatively selected in H-2bm13 animals persistently infected with the virus (neonatal virus carrier mice), nor could the transgenic TCR be activated by H-2bm13 infected cells in vivo or in vitro. These experiments show that although a TCR may be selected by a mutant MHC molecule, the corresponding viral antigen cannot be recognized in context of the mutant MHC molecule, as judged by both negative selection and T cell reactivity in vivo and in vitro. The 'enhanced' positive selection occurring in the context of Dbm13 suggests that a different conformation of the MHC molecule is able to select the same TCR and also that various TCR-ligand avidities may permit positive selection.

Amino Acid Sequence↗

PubMed1992

Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.

Base Sequence↗

PubMed2005

Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells.

An international collaborative project has been proposed to inactivate all mouse genes in embryonic stem (ES) cells using a combination of random and targeted insertional mutagenesis techniques. Random gene trapping will be the first choice in the initial phase, and gene-targeting experiments will then be carried out to individually knockout the remaining 'difficult-to-trap' genes. One of the most favored techniques of random insertional mutagenesis is promoter trapping, which only disrupts actively transcribed genes. Polyadenylation (poly-A) trapping, on the other hand, can capture a broader spectrum of genes including those not expressed in the target cells, but we noticed that it inevitably selects for the vector integration into the last introns of the trapped genes. Here, we present evidence that this remarkable skewing is caused by the degradation of a selectable-marker mRNA used for poly-A trapping via an mRNA-surveillance mechanism, nonsense-mediated mRNA decay (NMD). We also report the development of a novel poly-A-trap strategy, UPATrap, which suppresses NMD of the selectable-marker mRNA and permits the trapping of transcriptionally silent genes without a bias in the vector-integration site. We believe the UPATrap technology enables a simple and straightforward approach to the unbiased inactivation of all mouse genes in ES cells.

Animals↗

PubMed1991

Legislative change to permit direct access to physical therapy services: a study of process and content issues.

The purpose of this study was to examine process and content issues related to legislative change to permit direct access to physical therapy services. Data sources were survey questionnaires sent to the presidents of the 52 chapters of the American Physical Therapy Association (APTA), APTA publications, state statutes, and personal contacts. Results were based on the experiences of 35 chapters, 17 in direct-access states and 18 in non-direct-access states. The majority of direct-access states obtained their status in a single legislative campaign; the majority of non-direct-access jurisdictions attempting legislative change have been unsuccessful for 2 or more years. Over 80% of the chapters reported using legislative lobbyists. Opposing forces varied from state to state and included hospital and medical associations, physicians, chiropractors, and physical therapists. The following limitations on practice in a direct-access mode are found in the various practice acts: diagnosis requirements, eventual referral requirements, physical therapist qualifications, patient consent requirements, and practice setting restrictions.

Health Occupations↗

PubMed2001

The thalamus interrupts top-down attentional control for permitting exploratory shiftings to sensory signals.

When attention is involuntarily drawn in a direction different to that of the target, slower motor response times are observed (i.e. the meridian effect). Previous data suggested that the thalamus might participate in the generation of visual salience. What may be the role of the thalamus in the capture by luminance transients when attentional control is in action? A single experiment was administrated in a group of ten healthy volunteers as well as in a group of three patients with unilateral thalamic infarcts. Subjects participated in a task where attentional control was interrupted by a distractor. The meridian effect was present only in the performance of the healthy volunteers and when distractors occurred in the ipsilesional (intact) hemifield of the thalamic patients. These results suggest that when an important signal appears during attentional focalization, the thalamus interrupts current focalization and permits the compilation of an attentional program in the midbrain aiming at generating an orienting response towards the source of this signal.

Adult↗

PubMedDate not supplied

A novel percutaneous barrier device that permits safe subcutaneous access.

Successful subcutaneous access is important for optimal management of internal artificial organs and treatment of many diseases. However, skin downgrowth and tissue infection are still significant problems in the use of devices that require long-term subcutaneous access. The authors developed a new percutaneous device that provides a unique biologic boundary with surrounding connective tissue while minimizing the risk of complications. This new percutaneous device is made of a circumferential, strong, thin, and flexible mesh collar with millimeter size holes for connecting the structure. It has two distinct functional portions: a connecting zone and a sealing line. The most important features of the structure are: 1) Connective tissue grows through the millimeter size pores of the connecting zone to form a strong bond between the device and healthy tissue. 2) The millimeter size pores of the connecting zone allow capillaries to grow through its entire surface, ensuring sufficient blood supply. 3) The tissue of the connecting zone and the device surface form a biosealed junction (sealing line). This sealing line is protected by the connecting zone and is free from external forces acting on local skin. 4) The device is generally functional immediately after surgical implantation. The subcutaneous perimeter of the functional dome contains extruded rigid rings of millimeter size holes permitting growth of subcutaneous tissue through the device. This functions as the minor connecting structure of the device. Four of five implanted rabbits have remained healthy 8 months postimplantation without antibiotic administration. The fifth rabbit died 4 months postimplantation for reasons unrelated to the device. Gross or histopathologic inspection revealed no signs of tissue injury or inflammation. Growth of healthy connective tissue was clearly observed. These results indicate this percutaneous device can provide a strong, stable, and effective connection to internal organs without bioboundary damage, skin downgrowth, or tissue infection.

Animals↗

PubMed1999

Antihistamine prophylaxis permits rapid vancomycin infusion.

OBJECTIVE: To determine whether pretreatment with intravenous antihistamines attenuates the symptoms of red-man syndrome associated with rapid vancomycin administration. DESIGN: Prospective, randomized, double-blinded, placebo-controlled study of patients undergoing elective arthroplasty. SETTING: Preoperative unit in a tertiary care center. PATIENTS: Forty preoperative patients (American Society of Anesthesiologists status I-III, receiving vancomycin prophylaxis for elective prosthetic joint replacement or revision. INTERVENTIONS: Elective orthopedic patients were randomly allocated to receive intravenous antihistamines (diphenhydramine, 1 mg/kg, and cimetidine, 4 mg/kg) or placebo before rapid vancomycin infusion (1 g over 10 mins). Hemodynamic measurements, symptoms of histamine release, and plasma histamine levels were obtained in each patient during vancomycin administration. Rapid vancomycin infusion was discontinued in cases of decreases in mean blood pressure of > or =20% or intolerable itching. MEASUREMENTS AND MAIN RESULTS: Clinical symptomatology of red-man syndrome and histamine levels were assessed using Fisher's exact test or Student's t-test. Comparison of baseline and peak histamine levels for both the treated (mean +/- SD, 0.2 +/- 0.2 vs. 4.7 +/- 2.4 ng/mL; p < .0001) and placebo patients (mean +/-SD, 0.2 +/- 0.1 vs. 3.5 +/- 3.4 ng/mL; p = .0002) was statistically significant. Although there was a significant increase in plasma histamine levels during vancomycin infusion, it did not differ between the treatment groups. Only two (11%) of the treated patients developed hypotension, vs. 12 (63%) of the placebo patients (p = .002). Rash was partially attenuated. Twelve (63%) of the treated patients developed rash, compared with 19 (100%) of the placebo patients (p = .008). The rapid infusion was discontinued in two (11%) of the treated patients, compared with 11 (58%) of the placebo patients (p = .005). Four treated patients had no symptoms of histamine release. CONCLUSIONS: Pretreatment with intravenous H1 and H2 antihistamines permitted rapid vancomycin administration in 89% of treated patients. Although protection was incomplete, rash did not predict a need to stop the rapid infusion of vancomycin in our patients.

Adult↗

PubMed2004

Temporary intermaxillary fixation using individualized acrylic splints permits image-data-based surgery of the lower jaw and oropharynx.

OBJECTIVES/HYPOTHESIS: Image-data-based surgical navigation is used as a helpful device in the operating room to localize critical structures with a high degree of accuracy. It also enables physicians to plan therapeutic performance. Because it relies on preoperatively acquired computed tomography (CT) or magnetic resonance imaging (MRI) data, there is restricted access for navigation of surgical instruments in areas that show motion uncorrelated with radiologic data. Thus, in the case of moveable structures, for example the lower jaw, navigational procedures could not yet be applied. STUDY DESIGN: We introduce a new technique using individualized intermaxillary splints that fix the mandible in a reproducible aboccluded position at the time of image-data acquisition and surgery. METHODS: Different manufacturing processes were investigated. The feasibility of uni- and bilateral intermaxillary splints was studied under clinical conditions in four patients during different procedures in the mandibular and oropharyngeal regions. RESULTS: The manufacturing of the splints showed was easily performed in a short time. With bilateral fixation, there was a high anatomic target precision of 1.6 to 2.3 mm. CONCLUSIONS: The use of bilateral intermaxillary splints that fix the patient's mandible in a reproducible aboccluded position permits an image-data-based navigated surgical approach to the oropharyngeal and mandibular regions.

Humans↗

PubMed1990

The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirus genotypes.

A novel polymerase chain reaction (PCR) method was developed that permits the detection of 11 different human papillomavirus (HPV) genotypes using two general primer sets. By computer-assisted sequence analysis, two pairs of general primers were selected from the conserved L1 open reading frame and tested in the PCR on a set of cloned HPV genotypes. Experimental analysis showed that up to three mismatches between primers and target DNA did not influence the efficiency of the assay. The use of these primers in the PCR enabled the detection of HPV genotypes HPV-1a, -6, -8, -11, -13, -16, -18, -30, -31, -32 and -33, and was also successfully applied to well characterized cervical carcinoma cell lines and clinical samples. For the HPV types tested sub-picogram amounts of cloned DNA could be detected after general primer-mediated PCR and subsequent hybridization. The specificity of the amplification products was confirmed by blot hybridization procedures and RsaI restriction enzyme digestion. The results indicate that this PCR method can be a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas suspected of having an HPV aetiology.

Base Sequence↗

PubMed1994

Characterization of a Streptomyces-lividans-type site-specific DNA modification system in the avermectin-producer Streptomyces avermitilis permits investigation of two novel giant linear plasmids, pSA1 and pSA2.

The degradation of Streptomyces avermitilis DNA samples analysed by conventional pulsed-field gel electrophoresis was shown to be due to Tris-dependent, double-strand cleavage. Using alternative electrophoretic conditions, separation of intact DNA molecules was achieved, permitting the identification of two novel giant linear plasmids: the 100 kb pSA1 and 250 kb pSA2. Use of pSA2 DNA as a probe showed that pSA1 does not cross-hybridize, indicating that the plasmids are not closely related. The site-specificity of the DNA modifications, which render the DNA susceptible to Tris-dependent cleavage, was found to be essentially identical to that of similar modifications found in the DNA of S. lividans.

Bacterial Proteins↗

PubMed1994

Short-range repression permits multiple enhancers to function autonomously within a complex promoter.

Transcriptional repressors play a key role in establishing localized patterns of gene expression in the early Drosophila embryo. Several different modes of repression have been implicated in previous studies, including competition and direct interference with the transcription complex. Here, we present evidence for "quenching," whereby activators and repressors co-occupy neighboring sites in a target promoter, but the repressor blocks the ability of the activator to contact the transcription complex. This study centers on a zinc finger repressor, snail (sna), which represses the expression of neuroectodermal regulatory genes in the presumptive mesoderm. We show that sna can mediate efficient repression when bound 50-100 bp from upstream activator sites. Repression does not depend on proximity of sna-binding sites to the transcription initiation site. sna is not a dedicated repressor but, instead, appears to block disparate activators. We discuss the importance of quenching as a means of permitting separate enhancers to function autonomously within a complex promoter.

Animals↗

PubMed1999

A splice site mutant of maize activates cryptic splice sites, elicits intron inclusion and exon exclusion, and permits branch point elucidation.

DNA sequence analysis of the bt2-7503 mutant allele of the maize brittle-2 gene revealed a point mutation in the 5' terminal sequence of intron 3 changing GT to AT. This lesion completely abolishes use of this splice site, activates two cryptic splice sites, and alters the splicing pattern from extant splice sites. One activated donor site, located nine nt 5' to the normal splice donor site, begins with the dinucleotide GC. While non-consensus, this sequence still permits both trans-esterification reactions of pre-mRNA splicing. A second cryptic site located 23 nt 5' to the normal splice site and beginning with GA, undergoes the first trans-esterification reaction leading to lariat formation, but lacks the ability to participate in the second reaction. Accumulation of this splicing intermediate and use of an innovative reverse transcriptase-polymerase chain reaction technique (J. Vogel, R.H. Wolfgang, T. Borner [1997] Nucleic Acids Res 25: 2030-2031) led to the identification of 3' intron sequences needed for lariat formation. In most splicing reactions, neither cryptic site is recognized. Most mature transcripts include intron 3, while the second most frequent class lacks exon 3. Traditionally, the former class of transcripts is taken as evidence for the intron definition of splicing, while the latter class has given credence to the exon definition of splicing.

Alternative Splicing↗

PubMed1994

Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome-binding site permits tap-independent RepA synthesis.

The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher repA expression. In Incl alpha/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.

Bacterial Proteins↗

PubMed1985

Expression in mammalian cells of the diaminopimelic acid decarboxylase of Escherichia coli permits cell growth in lysine-free medium.

The lysA gene of Escherichia coli encodes for a diaminopimelic acid decarboxylase (EC 4.1.1.20) which allows the conversion of diaminopimelic acid into lysine in bacteria. It has been cloned in an eukaryotic expression vector containing upstream the SV40 early promoting sequence, and downstream mouse alpha-globin maturating sequences. The recombinant plasmid pSB99 (4800 base pairs) has been introduced into several mammalian cell lines by cotransfection with a second selectable marker i.e. the polyoma-transforming DNA. Selection for morphologically transformed rat cells which contained the intact lysA sequences, allowed the determination of the concentration of diaminopimelic acid in the lysine-free medium that permitted cell growth. lysA-expressing clones were directly selected in a medium containing 10 mM diaminopimelic acid, after transfection with pSB99 alone. Southern blot analysis on selected clones have shown that they contain up to 30-50 integrated copies of the plasmid in tandem arrangement. Finally, we demonstrated that lysA-expressing clones incorporate a significant amount of radiolabelled [3H]diaminopimelic acid in acid-insoluble material. The recombinant plasmid can serve as a selectable marker, in growth medium in which lysine was replaced by its direct bacterial precursor.

Animals↗