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At least 343 records · Page 19Linked to original sources
PubMed1985

Genetic analysis of spontaneous resistance to ampicillin in Neisseria gonorrhoeae.

Step-wise intrinsic resistance to ampicillin in Neisseria gonorrhoeae was analyzed genetically by DNA-mediated transformation experiments. A first-step ampicillin-resistant (Ampr1) mutant and a second-step ampicillin-resistant (Ampr2) mutant generated during sequential selection were used in these studies. Each selection step was accompanied by an approximate twofold increase in resistance. Four amp alleles were found to account for full resistance of the Ampr2 phenotype. All four amp alleles lie among a cluster of genes which code for ribosomal functions. This region has the map order rif str fus tet cam. First-step resistance was caused by two amp alleles, ampA2 and ampB1, neither of which independently caused detectable ampicillin resistance. Outcrossing of the ampA2 or the ampB1 mutation resulted in wild-type susceptibility to ampicillin. Mapping studies indicate that ampB1 lies between str and fus, whereas ampA2 lies to the right of cam. Second-step resistance required two mutations, ampC3 and ampD4, in addition to ampB1 and ampA2. Transformation of ampC3 to ampC3+ in an Ampr2 mutant resulted in the Ampr1 phenotype. Both ampC3 and ampD4 showed transformation linkage to rif and str. ampC3 was positioned at a site between rif and str. ampD4 apparently occupied a site, outside of the rif-str region, proximal to rif and distal to str. We postulate the gene order to be ampD rif ampC str ampB fus tet cam ampA.

Alleles↗
PubMed1984

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

Asparagine↗
PubMedDate not supplied

Cytoskeleton and epithelial polarity.

The membrane surface of polarized epithelial cells can be divided in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton are involved in critical steps of both generation and maintenance of cell polarity. Generation of polarity is controlled by microtubules that serve as uniformly aligned and polarized cytoplasmic guiding structures for the vectorial and selective transport of Golgi-derived carrier vesicles to the apical cell surface. Targeting of membrane proteins to the basolateral cell surface does not depend on microtubules but follows the constitutive bulk flow of membranes. Once inserted into the lipid bilayer several membrane proteins such as the kidney anion exchanger 1 (AE1) and the sodium pump become immobilized at specialized microdomains of the lateral cell surface. Evidence is provided that both membrane proteins are linked via ankyrin to the spectrin-based membrane cytoskeleton that underlies the basolateral membrane domain. Linkage of these and other integral membrane proteins to the cytoskeleton may not only place them to specialized sites of the plasma membrane but may also prevent these transporters from clustering and endocytosis, thus helping them to stay at the cell surface. In search of sequence motifs involved in binding of integral membrane proteins to components of the cytoskeleton we found that the binding interface of AE1 to protein 4.1 (an actin and spectrin cross-linking protein) consists of a cluster of five amino acid residues, namely IRRRY in AE1 and LEEDY on protein 4.1. This motif may play a more general role in cytoskeleton membrane linkages.

Amino Acid Sequence↗
PubMed1998

Comparison of performance-based and patient-reported measures of function in anterior-cruciate-ligament-deficient individuals.

There is a dearth of reliable and valid instrumentation that measures disability following injury and/or surgery of the knee joint that is responsive to clinically significant changes over time. The purpose of this investigation was to determine whether performance-based or patient-reported measures of function are more effective in estimating disability in individuals with an anterior-cruciate-ligament (ACL)-deficient knee. Subjective rating of knee function was used as the criterion measure for disability, and selected performance-based and patient-reported measures were used as estimation variables. Twenty-nine individuals with an ACL-deficient knee participated in this investigation. Step-wise regression analysis revealed that the Cincinnati Knee Scale, Lysholm Knee Scale, and hop index were the most effective estimates of disability. The results demonstrate that patient-reported measures are more related to the patient's level of disability in individuals with an ACL-deficient knee. More research is necessary to substantiate these findings.

Adolescent↗
PubMed1985

[Current in vivo and in vitro diagnosis of thyroid diseases].

A single method which is sufficient to meet all diagnostic requirements in patients with thyroid disorders does not exist. In selecting a test combination one should always consider, that differentiation of patients with hyper- or hypothyroidism from those with euthyroidism should involve the least effort. A meaningful step-by-step diagnostic work-up should always start with individually selected in vitro tests, followed by in vivo methods such as ultrasonography, radionuclide scanning and X-ray examination. This accords with the recommendations for diagnosis of disturbed thyroid function and thyroid disorders published recently by the thyroid section of the German Association of Endocrinology.

Humans↗
PubMed1985

Characterization of a Euglena gracilis chloroplast RNA polymerase specific for ribosomal RNA genes.

Euglena gracilis chloroplasts contain a 145,000-base pair chromosome that encodes genes for ribosomal, transfer, and messenger RNAs. These genes are transcribed within the organelle by chloroplast RNA polymerase activities that are specific for different classes of RNA. Two transcriptional activities have been isolated from Euglena chloroplasts. (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259, 14880-14887). One, the "soluble extract," contains enzymes active in tRNA transcription and processing. The other activity, the transcriptionally active chromosome, consisting of a chloroplast DNA-dependent RNA polymerase tightly bound to chloroplast DNA, only transcribes rRNA genes even though the entire chloroplast genome is present. We have extensively purified the transcriptionally active chromosome using high salt concentrations to dissociate loosely bound proteins. The result is a highly enriched extract containing three major polypeptides of Mr 116,000-118,000, 83,000-88,000, and 24,000-26,000 that retains complete selectivity for rDNA transcription. It is probable that one, or both, of the high molecular weight proteins are functional components of the DNA-dependent RNA polymerase. The identification and characterization of the transcriptionally active chromosome is a first step towards understanding how chloroplast rRNA synthesis is regulated.

Chloroplasts↗
PubMedDate not supplied

Modulation of human immune responsiveness in vitro by auranofin.

The effect of auranofin (AF) on in vitro correlates of human immune responsiveness was examined. AF inhibited mitogen induced human lymphocyte proliferation and the generation of immunoglobulin secreting cells in a concentration dependent manner. The inhibition was most effective when AF was present from the initiation of culture indicating that this drug blocked a critical early step in lymphocyte activation. Marked inhibition of mitogen responsiveness was observed as a result of a 1-h preincubation with AF. The brief preincubation with low concentrations of AF (0.3 micrograms/ml) resulted in a selective inhibition of the accessory function of monocytes but had no effect on potential lymphocyte responsiveness. Preincubation with higher concentrations of AF (greater than 0.6 micrograms/ml) resulted in a more non-specific inhibition of both monocyte and lymphocyte function. These data support the conclusion that AF may function as an immunosuppressive agent.

Antibody Formation↗
PubMedDate not supplied

[The interneuronal functional connections in the sensorimotor cortex of dogs].

Multiunit activity of sensorimotor cortex was recorded from chronically implanted semi-microelectrodes in two dogs. Functional interneuronal connections between neuronal spike trains of 6-8 neurons selected from background multiunit activity were studied by the method of cross-correlation analysis. Bin widths 0.5, 1, 2, 3 and further up to 40 ms by step of 1 ms were used. The cross-interval connections were characterized by complete absence of the shared input (central symmetrical peaks) and signs of inhibitory interrelations. The temporal interrelations between selected neurons were characterized by unilateral and bilateral non-symmetrical excitatory connections--ultra-narrow peaks with short (1-10 ms), middle (10-80 ms) and long (80-2000 ms) delays. The existence of such ultra-narrow peaks contradicts "classical" conceptions on the character of cross-interval connections based on model experiments on simple nervous systems. We suppose that special mechanism of synchronization with high temporal accuracy exists in the cortex.

Action Potentials↗
PubMed1995

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique.

The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Animals↗
PubMed1991

Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.

Amino Acid Sequence↗
PubMed1995

Interstitial lung diseases in children: a review.

Interstitial lung diseases (ILD) are disorders of the lower respiratory tract, characterized by chronic inflammation of the lung parenchyma, varying degree of fibrosis, derangement of the alveolar walls and loss of the functional alveolar capillary units. ILD are relatively uncommon in children. Most of the interstitial lung diseases have no known etiology. In children, common diseases associated with ILD include viral respiratory tract infections (RSV, parainfluenza, etc.), gastroesophageal reflux, idiopathic pulmonary fibrosis, pulmonary hemosiderosis, eosinophilic pneumonia, pneumonitis associated with AIDS, etc. Chronic inflammation of the alveoli (alveolitis), the initial injury in ILD, and several mediators released from inflammatory cells (eosinophils, neutrophils and macrophages) can cause fibrosis and derangement of alveolar walls. Dyspnea and a non-productive cough are the cardinal symptoms of ILD. Other findings include chest pain, hemoptysis and weight loss. Clubbing of fingers occur in approximately 50 per cent of cases. Diagnosis is based on a combination of history, clinical findings, radiographic findings, pulmonary function tests and histologic findings. Open lung biopsy has been very helpful in providing information regarding the extent and nature of the damage, prognosis and response to therapy. There are 3 main aspects in the treatment of ILD. The most important step is to identify and eliminate the cause. The second is suppression of the inflammation. The third is supportive and symptomatic treatment. Corticosteroids are the drugs commonly used for suppression of inflammation. Immunosuppressive drugs (azathioprine, cyclophosphamide) have also been tried. Lung transplantation and heart transplantation have been successfully achieved in selected patients. The results of therapy should be regularly monitored by clinical symptoms, chest radiographs and serial pulmonary function studies.

Age of Onset↗
PubMed1998

Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells.

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, approximately 0. 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT-PCR, demonstrating that sequences contained on the BAC are expressed. We believe that GFP-neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.

Animals↗
PubMed1995

Single-step purification of prostatic acid phosphatase: immunoaffinity chromatography with a monoclonal antibody.

BACKGROUND: Prostatic acid phosphatase (PAP) is an important protein which should be studied further as a tumor marker or as a biologically functional molecule. The purpose of the study was to establish a simple and reliable method to obtain highly pure PAP. METHODS: Spleen cells from mice immunized with prostatic epithelial cells prepared from benign prostatic hyperplasia tissue were fused with myeloma cells X63Ag8-653. Hybrid cells of interest were selected using the indirect immunofluorescence method with unfixed frozen tissue sections. One clone of the hybrid cell lines was established which secreted the monoclonal antibody specifically reactive to prostatic acid phosphatase. Using this monoclonal antibody, we purified the antigen from human prostatic tissue by means of single-step immunoaffinity chromatography. RESULTS: SDS-PAGE profiling under reducing conditions indicated that the protein recognized by this antibody consisted of several components of molecular weight 41,000-45,000. Partial amino acid sequence analysis of this protein indicated that these components involved a heterogeneously modified single polypeptide, and that this antigen is identical to human prostatic acid phosphatase. CONCLUSIONS: This single-step method saves the time needed to purify prostatic acid phosphatase and requires only half a day for the whole procedure. Moreover, the purity of the isolated protein was extremely high. This method seems to be useful not only for purifying prostatic acid phosphatase but also for purifying other proteins from the prostate gland and for analysis of antigenic macromolecules.

Acid Phosphatase↗
PubMed1993

Measles virus-induced changes in leukocyte function antigen 1 expression and leukocyte aggregation: possible role in measles virus pathogenesis.

Measles virus (MV) infection of U937 cell or peripheral blood leukocyte cultures was shown to induce changes in the expression of leukocyte function antigen 1 (LFA-1) and cause marked aggregation of these cells. Addition of selected monoclonal antibodies specific for LFA-1 epitopes that did not neutralize MV in standard neutralization assays were found to block both virus-induced leukocyte aggregation and virus dissemination. These data suggest that MV modulation of LFA-1 expression on leukocytes may be an important step in MV pathogenesis.

Antibodies, Monoclonal↗
PubMed1983

Quantitative profiling of prostaglandins and thromboxane by high-resolution gas chromatography-selected--ion monitoring.

The development and biological application of a rapid method for quantitative profiling of prostaglandins and thromboxane using high-resolution gas chromatography (HRGC) coupled with mass spectrometry in the selected-ion monitoring technique (SIM) are described. The method is based on the single-step extraction of prostaglandins from biological samples on C18 reversed-phase cartridges after addition of deuterated analogues as internal standards, followed by derivatization of functional groups and final analysis by HRGC-SIM with wall-coated open tubular persilanized capillary columns. Biological applications include the determination of endogenous arachidonic acid cascade profiles in rat tissue homogenates and thromboxane synthetase inhibition studies in human serum.

6-Ketoprostaglandin F1 alpha↗
PubMed1999

Making artificial antibodies: a format for phage display of combinatorial heterodimeric arrays.

The gene VII protein (pVII) and gene IX protein (pIX) are associated closely on the surface of filamentous bacteriophage that is opposite of the end harboring the widely exploited pIII protein. We developed a phagemid format wherein antibody heavy- and light-chain variable regions were fused to the amino termini of pVII and pIX, respectively. Significantly, the fusion proteins interacted to form a functional Fv-binding domain on the phage surface. Our approach will be applicable to the display of generic peptide and protein libraries that can form combinatorial heterodimeric arrays. Consequently, it represents a first step toward artificial antibodies and the selection of novel biological activities.

Amino Acid Sequence↗
PubMed1976

Fast and slow pyramidal tract neurons: an intracellular analysis of their contrasting repetitive firing properties in the cat.

1. Intracellular recordings were made from an estimated 500 neurons in the sensorimotor cortex of barbiturate-anesthetized cats. Of those which were antidromically identified from the medullary pyramids, 70 were selected which also exhibited steady repetitive firing to steps of current injected through the recording electrode; 81% were "fast" (conduction velocity greater than 20 m/s) and 19% were "slow". 2. As shown by earlier workers, the spike duration is a function of conduction velocity; a spike duration of 1.0 ms is the dividing line between fast and slow. 3. Of the 57 fast pyramidal tract neurons (PTNS), 14 exhibited double spikes during otherwise rhythmic firing patterns to a step of injected current. These very short interspike intervals (usually 1.5-2.5 ms) were first seen interspersed in a rhythmic discharge (e.g., 50-ms intervals) but, with further increases in current strength, would come to dominate the firing pattern; e.g., double spikes every 40 ms. Further increases in current would typically shorten only the long intervals; e.g., 40-30 ms, but some fast PTNS developed triple spikes, etc. 4. The extra spike appears to arise from a large hump which follows most spikes in fast PTNS; while this humplike "depolarising after-potential" can also be seen in slow PTNS, it is small. Extra spikes were seen only in fast PTNS with large postspike humps; in perhaps half of the fast PTNS, extra spikes probably contributed to "adaptation." 5. Slow PTNS often had frequency-current curves which were not repeatable; a "hysteresis" phenomenon could often be seen, where the proportionality constant relating current to firing rate decreased following high firing rates. 6. The B spike was distinguishable from the A spike in differentiated antidromic spikes in 77% of the slow PTNS, in only 14% of the fast PTNS which later exhibited double spikes during current-induced repetitive firing, and in 53% of the other fast PTNS. 7. The antidromic spike heights of doublet PTNS were not significantly different from those of other repetitively firing PTNS.

Animals↗
PubMed1995

Functional desensitization of beta agonist responses in human lung mast cells.

The beta adrenergic agonist isoprenaline inhibited the IgE-triggered release of the preformed mediator histamine from human lung mast cells (HLMC) in a dose-dependent fashion. After prolonged (> or = 4 h) preexposure of HLMC to isoprenaline, there was a subsequent diminution in the effectiveness of a second exposure of isoprenaline to inhibit the release of histamine from activated HLMC. This induced hyporesponsiveness to isoprenaline was both concentration and time dependent. Although maximal levels of desensitization were obtained after an initial prolonged (14-h) preincubation with a high (10(-5) M) concentration of isoprenaline, exposure of HLMC for a shorter (4-h) time period with a lower (3 x 10(-7) M) concentration of isoprenaline was also effective at inducing a functional desensitization to isoprenaline. The inhibitory activity of the beta 2 agonist fenoterol was attenuated after a prolonged (14-h) pretreatment step with isoprenaline (10(-5)M), whereas the inhibitory properties of other adenylate cyclase activators, prostaglandin E2 and forskolin, were not affected appreciably. Prolonged (12-h) exposure of HLMC to the beta agonists fenoterol, salbutamol, and terbutaline also induced hyporesponsive states of beta agonists, qualitatively similar to that obtained with isoprenaline. The beta receptor antagonist propranolol, if coincubated with isoprenaline during the prolonged pretreatment step, protected against the subsequent refractoriness of the HLMC to isoprenaline. The glucocorticoid dexamethasone failed to prevent the isoprenaline-induced functional desensitization. In total, these results indicate that prolonged exposure of HLMC to beta agonists induces a state of selective hyporesponsiveness to agonists that act at beta adrenoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗