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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↗
PubMed1999

Total Synthesis of (+)-Halichlorine: An Inhibitor of VCAM-1 Expression.

The diastereoselective addition of the highly functionalized organozinc compound 1 to the aldehyde 2 in the presence of the chiral amino alcohol 3 (-->4) is a key step in the first total synthesis of (+)-halichlorine. A series of protections/deprotections and a macrolaconization complete the synthesis. Halichlorine selectively inhibits the expression of the cell adhesion molecule VCAM-1. TBS=tert-butyldimethylsilyl.

Journal Article↗
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↗
PubMed1976

A spin label study of the thermal unfolding of secondary and tertiary structure in E. colic transfer RNAs.

The molecular mechanism of thermal unfolding of E. coli tRNAGlu, tRNAfMet and tRNAPhe (in 0.02M Tris-HC1, pH 7.5. 10 MM Mg C12) has been examined by the spin-labeling technique. The rate of tumbling of the spin label has been measured as a function of temperature for ten different selectively spin-labeled tRNAs. Only spin labels at position s4U-8 were able to probe the tertiary structure. Evidences are presented which support the hypothesis that the thermal denaturation of the three species of tRNAs studied is sequential. The unfolding process occurs in three discrete stages. The first step (30 degrees-32 degrees) could either be assigned to a localized reorganization of the cold-denatured structure or to a "transient" melting, followed by the simultaneous disruption of the tertiary structure and part of the hU helix. This transition is observed even in the absence of magnesium. The second step (50 degrees-54 degrees) involves the melting of the anticodon and miniloop regions. The last step occurs above 65 degrees where the t psi c and amino acid acceptor stems, forming one continuous double helix, melt. A simple dynamic model is considered for tRNA function in protein biosynthesis.

Binding Sites↗
PubMed1998

Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.

In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre-mRNA splicing. Prp22 can act on pre-assembled spliceosomes that are arrested after step 1 in an ATP-independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3' splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast pre-mRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome.

Actins↗
PubMed1995

AF150(S): a new functionally selective M1 agonist improves cognitive performance in rats.

This study was aimed at evaluating the ability of a new functionally selective partial M1 agonist, AF150(S), to reverse cognitive impairments in rats. A memory deficits-induced animal model was used that involved AF64A (3 nmol/2 microliters/side) bilaterally injected ICV. AF150(S) was administered PO. The pharmacodynamic profile of the compound was established and its general toxicity was evaluated. Animals were tested on three behavioral tasks: step-through passive avoidance, Morris water maze reference memory paradigm, and radial arm maze working memory paradigm. The sign-free dose of AF150(S) was > 40 mg/kg whereas the LD50 was > 500 mg/kg. In comparison, the effective dose in reversing performance impairments on the various tasks was much lower (0.5-5 mg/kg). The data suggest that AF150(S) possesses potential cognitive enhancement abilities, probably due to a specific increase of cholinergic function.

Animals↗
PubMed1998

Rab11 is required for trans-golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor.

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum-to-Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

Animals↗
PubMed1989

Detection of temporal gaps in sinusoids by normally hearing and hearing-impaired subjects.

A two-alternative forced-choice task was used to measure psychometric functions for the detection of temporal gaps in a 1-kHz, 400-ms sinusoidal signal. The signal always started and finished at a positive-going zero crossing, and the gap duration was varied from 0.5 to 6.0 ms in 0.5-ms steps. The signal level was 80 dB SPL, and a spectrally shaped noise was used to mask splatter associated with the abrupt onset and offset of the signal. Two subjects with normal hearing, two subjects with unilateral cochlear hearing loss, and two subjects with bilateral cochlear hearing loss were tested. The impaired ears had confirmed reductions in frequency selectivity at 1 kHz. For the normal ears, the psychometric functions were nonmonotonic, showing minima for gap durations corresponding to integer multiples of the signal period (n ms, where n is a positive integer) and maxima for durations corresponding to (n - 0.5) ms. For the impaired ears, the psychometric functions showed only small (nonsignificant) nonmonotonicities. Performance overall was slightly worse for the impaired than for the normal ears. The main features of the results could be accounted for using a model consisting of a bandpass filter (the auditory filter), a square-law device, and a sliding temporal integrator. Consistent with the data, the model demonstrates that, although a broader auditory filter has a faster transient response, this does not necessarily lead to improved performance in a gap detection task. The model also indicates that gap thresholds do not provide a direct measure of temporal resolution, since they depend at least partly on intensity resolution.

Aged↗
PubMed1999

Tuning biphenyl dioxygenase for extended substrate specificity.

Highly substituted polychlorinated biphenyls (PCBs) are known to be very resistant to aerobic biodegradation, particularly the initial attack by biphenyl dioxygenase. Functional evolution of the substrate specificity of biphenyl dioxygenase was demonstrated by DNA shuffling and staggered extension process (StEP) of the bphA gene coding for the large subunit of biphenyl dioxygenase. Several variants with an extended substrate range for PCBs were selected. In contrast to the parental biphenyl dioxygenases from Burkholderia cepacia LB400 and Pseudomonas pseudoalcaligenes KF707, which preferentially recognize either ortho- (LB400) or para- (KF707) substituted PCBs, several variants degraded both congeners to about the same extent. These variants also exhibited superior degradation capabilities toward several tetra- and pentachlorinated PCBs as well as commercial PCB mixtures, such as Aroclor 1242 or Aroclor 1254. Sequence analysis confirmed that most variants contained at least four to six template switches. All desired variants contained the Thr335Ala and Phe336Ile substitutions confirming the importance of this critical region in substrate specificity. These results suggest that the block-exchange nature of gene shuffling between a diverse class of dioxygenases may be the most useful approach for breeding novel dioxygenases for PCB degradation in the desired direction.

Amino Acid Sequence↗
PubMedDate not supplied

Construction of Zn2+/Cd(2+)-tolerant cyanobacteria with a modified metallothionein divergon: further analysis of the function and regulation of smt.

This paper reports the (de novo) construction of mutants of Synechococcus PCC 7942 lacking the repressor (SmtB) of the metallothionein gene, smtA. These smtA+/B- cells are more tolerant to elevated [Zn2+] and [Cd2+] than cells containing an intact metallothionein divergon (smt). Previously selected (by step-wise adaptation) Cd(2+)-tolerant mutants contain additional copies of smtA and possibly other undetected mutations. It is now confirmed that these cells also contain a deletion within 'all' copies of smtB and hence fail to revert to wild type following subculture in medium which has not been supplemented with Cd2+ or Zn2+. Northern analysis showed enhanced accumulation of smtA transcripts, even in the absence of added metal ions in these mutants. An increase in the accumulation of Zn2+ is reported in cells containing an intact metallothionein divergon compared to cells deficient in both smtA and smtB. This supports the assumption that SmtA binds Zn2+ within cyanobacterial cells. We also describe the use of the above mentioned mutants to identify additional factors involved in the regulation of transcription from the smtA operator-promoter.

Bacterial Proteins↗