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At least 487 records · Page 27Linked to original sources
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↗
PubMed2001

Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+ concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells.

On reaching the oviduct, spermatozoa are retained in the isthmic region of the oviduct until ovulation occurs. The essential steps of capacitation are co-ordinated in this region. In this study, a primary cell culture system of oviductal epithelial cells was established to investigate sperm binding to oviductal epithelium and modulation of sperm function during incubation under capacitating conditions in co-culture with oviductal epithelial cells. Epithelial cells were stripped from the oviducts of sows and cultivated for 5-7 days on Lab-Tek Chamber slides on Matrigel. The preparations on chamber slides and suspensions of control spermatozoa were incubated for 3 h in Tyrode's albumin lactate pyruvate (TALP) medium. At 3, 30, 60, 90 and 180 min the free-swimming spermatozoa were collected by washing, and membrane integrity, tyrosine phosphorylation patterns and [Ca(2+)](i) of bound, unbound and control spermatozoa were assessed with fluorescent probes (propidium iodide, Cy-3 and fluo-3-AM). The cells bound to oviductal epithelial cells showed reduced cytosolic Ca(2+) concentration, reduced and almost absent tyrosine phosphorylation of membrane proteins and higher viability at the time of the first sampling. Increases in Ca(2+) concentration and cell death occurred much more slowly during incubation in cells bound to oviductal epithelial cells compared with free-swimming spermatozoa, and no changes in tyrosine phosphorylation were observed. The preferential binding of viable, low-Ca(2+) cells with suppressed tyrosine phosphorylation and slower functional modulation of boar spermatozoa attached to oviductal epithelial cells might represent a mechanism for selecting functionally competent spermatozoa and prolonging their lifespan by delaying capacitation in the oviductal reservoir.

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↗
PubMed1996

Synthesis and Properties of Substituted CBI Analogs of CC-1065 and the Duocarmycins Incorporating the 7-Methoxy-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (MCBI) Alkylation Subunit: Magnitude of Electronic Effects on the Functional Reactivity.

The synthesis of 7-methoxy-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (MCBI), a substituted CBI derivative bearing a C7 methoxy group para to the C4 carbonyl, is described in efforts that establish the magnitude of potential electronic effects on the chemical and functional reactivity of the agents. The core structure of the MCBI alkylation subunit was prepared by a modified Stobbe condensation/Friedel-Crafts acylation for generation of the appropriately functionalized naphthalene precursors (15 and 20) followed by 5-exo-trig aryl radical-alkene cyclization (24 --> 25, 32 --> 33) for completion of the synthesis of the 1,2-dihydro-3H-benz[e]indole skeleton and final Ar-3' alkylation of 28 for introduction of the activated cyclopropane. Two approaches to the implementation of the key 5-exo-trig free radical cyclization are detailed with the former proceeding with closure of 24 to provide 25 in which the required product functionalization was introduced prior to cyclization and the latter with Tempo trap of the cyclization product of the unfunctionalized alkene substrate 32 to provide 33. The latter concise approach provided the MCBI subunit and its immediate precursor in 12-13 steps in superb overall conversions (27-30%). Resolution of an immediate MCBI precursor and its incorporation into both enantiomers of 39-46, analogs of CC-1065 and the duocarmycins, are detailed. A study of the solvolysis reactivity and regioselectivity of N-BOC-MCBI (29) revealed that introduction of the C7 methoxy group accelerates the rate of solvolysis by only 1.2-1.06x. This remarkably modest effect is inconsistent with C4 carbonyl protonation as the slow and rate-determining step of solvolysis or acid-catalyzed nucleophilic addition but is consistent with a mechanism in which protonation is rapid and reversible followed by slow and rate-determining nucleophilic addition to the cyclopropane requiring both the presence and assistance of a nucleophile (S(N)2 mechanism). No doubt this contributes to the DNA alkylation selectivity of this class of agents and suggests that the positioning of an accessible nucleophile (adenine N3) and not C4 carbonyl protonation is the rate-determining step controlling the sequence selectivity of the DNA alkylation reaction. This small electronic effect on the solvolysis rate had no impact on the solvolysis regioselectivity, and stereoelectronically-controlled nucleophilic addition to the least substituted carbon of the activated cyclopropane was observed exclusively. For the natural enantiomers, this unusually small electronic effect on functional reactivity had little or no perceptible effect on their DNA alkylation selectivity, efficiency, and relative rates or on their biological properties. Perceptible effects of the C7 methoxy substituent on the unnatural enantiomers were observed and they proved to be 4-40x more effective than the corresponding CBI-based unnatural enantiomers and comparable in cytotoxic potency with the MCBI natural enantiomers. This effect is most consistently rationalized not by a C7 methoxy substituent effect on functional reactivity but rather through introduction of additional stabilizing noncovalent interactions which increase the unnatural enantiomer DNA alkylation efficiency and further stabilize its inherently reversible DNA alkylation reaction.

Journal Article↗
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↗
PubMed1978

Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene. Cross-link localization.

At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the epsilon-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 A (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5'-phosphate in the active site.

Animals↗
PubMed1989

Localization of cutaneous lesions in digital images.

Digital imaging could potentially provide a rapid, objective, and quantitative means of detecting changes in important skin conditions, especially the dysplasic nevus syndrome. Image analysis techniques can be applied to digital images to automate the search for changes in moles or other features. Consistent determination of lesion boundaries, perimeter, and area in digital images is a vital first step in this process. In this paper, we show how bilaterally symmetric Laplacian-of-a-Gaussian filters can be used to recover the borders of selected lesions while remaining robust with respect to factors such as the camera point spread function and additive noise. Tests on real and synthetic images demonstrate that lesion borders, area, and perimeter can be obtained with a high degree of reliability. Boundaries are routinely found to within +/- 0.2 pixels, and area and perimeter measurements vary by less than 10% when imaging spot targets and actual cutaneous lesions under a realistic range of experimental conditions.

Dysplastic Nevus Syndrome↗
PubMed1983

Teleonomical optimization of a fractal model of the pulmonary arterial bed.

Modeling the pulmonary arterial tree (PAT) is considered here as an optimal synthesis problem. Firstly, a class of candidate models is specified: the three-dimensional symmetric dichotomous fractal trees of elastic tubes described by Womersley's equations. Secondly, the parameters are shown to be constrained by interactions of PAT with the rest of the body; these constraints are used to limit the volume of the parametric space to which attention will be directed in the synthesis step. Thirdly, a teleonomical hypothesis is proposed: a naturally selected PAT must have a minimal input impedance under conditions keeping total arterial volume and distensibility as small as possible. This hypothesis is translated in mathematical terms and the resulting cost-function minimized in the limited parametric volume. The optimal model has parameter values and an impedance spectrum corresponding satisfactorily with real data. Moreover this model gives a clear picture of the internal hemodynamic behavior of PAT as an impedance matching device.

Blood Flow Velocity↗
PubMed1999

A novel human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates.

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase gene family, designated GalNAc-T7, was cloned and expressed. GalNAc-T7 exhibited different properties compared to other characterized members of this gene family, in showing apparent exclusive specificity for partially GalNAc-glycosylated acceptor substrates. GalNAc-T7 showed no activity with a large panel of non-glycosylated peptides, but was selectively activated by partial GalNAc glycosylation of peptide substrates derived from the tandem repeats of human MUC2 and rat submaxillary gland mucin. The function of GalNAc-T7 is suggested to be as a follow-up enzyme in the initiation step of O-glycosylation.

Amino Acid Sequence↗
PubMed2000

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway.

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.

Animals↗
PubMed1988

In vivo stimulation and restoration of the immune response by the noninflammatory fragment 163-171 of human interleukin 1 beta.

The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, showed in vivo immunomodulatory capacities qualitatively and quantitatively comparable to those of the mature human IL-1 beta protein. In fact, both IL-1 beta and the 163-171 fragment stimulated the immune response of normal mice and restored immune reactivities of immunocompromised animals. In addition, the synthetic IL-1 peptide was as efficient as the entire protein in inducing tumor rejection and radioprotection. On the other hand, the 163-171 fragment did not cause any of several inflammation-associated metabolic changes inducible by the whole IL-1 beta molecule in vivo: hypoferremia, hypoglycemia, hyperinsulinemia, increase in circulating corticosterone, SAA and fibrinogen, decrease in hepatic drug-metabolizing enzymes. Furthermore, at variance with IL-1 beta, the 163-171 peptide did not show the toxic effects causing shock and death in adrenalectomized mice. Thus, these results confirm our previous in vitro observations that functional domains are identifiable within the multipotent cytokine IL-1 beta, and demonstrate the biological relevance of this finding in a variety of in vivo systems. The identification of a selectively active fragment of a cytokine may thus represent a significant step towards a better directed and more rational immunotherapeutic approach.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1998

Selective vulnerability of the developing brain to lead.

Environmental lead exposure in young children who ingest household paint dust or other sources impairs their potential intelligence in a linear, dose-dependent fashion in contrast to its far more subtle effects on other neurologic functions. Basic investigations have identified three interrelated steps in synaptic neurotransmission at which low levels of lead can disrupt signal processing. Lead enhances background transmitter release, but impairs stimulated release, inhibits function at the N-methyl-D-aspartate-type glutamate receptor and stimulates background levels of the intracellular messenger protein kinase C. Taken together these effects have the effect of diminishing the synaptic signal to noise ratio. The ability of lead to enhance 'synaptic noise' during a critical early period of postnatal development may permanently disrupt the architecture of cortical processing units by depriving them of high resolution environmental signals needed to refine synaptic connections.

Adolescent↗
PubMed1987

Patterns of outpatient consultation: a survey of outpatients attending a metropolitan psychiatric hospital.

A survey of all outpatients (n = 1135) attending a regional psychiatric hospital during a 3-month period. Psychoses constituted 72% and non-psychotic disorders 25%. Psychoses had a greater proportion of long-term attenders while non-psychotic disorders, though frequent (43%) among recent attenders, showed a high attrition rate. Demographic characteristics, patterns of attendance, selected clinical features, level of functioning and difficulties of management of the different diagnostic groups are presented and discussed. Reasons are adduced to explain why patients who were deemed suitable for transfer to other care had not been discharged from hospital. Steps taken to reduce the patient load and increase the efficiency of the services are described.

Adolescent↗
PubMed1996

UV photoaffinity labeling of Tn3 transposase--DNA complexes: identification of DNA binding domains.

The prokaryotic transposon Tn3 requires the transposase protein, as well as the cis-acting terminal inverted repeats (IRs), for transposition. The first step in the transposition process requires transposase binding to the IRs, as well as target site selection for element insertion. The primary aim of this study is to define the relationship between the structure of Tn3 transposase and its DNA binding functions. We have defined, by UV cross-linking, two broad regions of transposase that interact with DNA: a 70-kDa N-terminal domain and a 30-kDa C-terminal domain. The 70-kDa N-terminal domain encompasses the IR sequence specific binding domain, as well as a nonspecific DNA binding domain that has been previously described. We have also defined, by UV cross-linking, a region in the nonspecific DNA binding domain centered at amino acids 376 and 381 that is in contact with DNA. We have used site-directed mutagenesis of amino acids 376 and 381 to help delineate the function of this region of the transposase protein. Mutations in this region reduce transposition frequency to 30-40% of the wild type. These mutations reduce nonspecific DNA binding three- to four-fold but do not appear to affect specific binding to the IR. Transposition immunity is unaffected by mutations in the nonspecific DNA binding domain. This suggests that this region may be involved in target site selection.

Affinity Labels↗
PubMed1983

Age, sex, and body composition as predictors of children's performance on basic motor abilities and health-related fitness items.

The purpose of this study was to determine the contribution of age, sex, and body composition to children's motor performances on selected basic motor tasks, balance, speed, agility, power, coordination, and reaction time, and health-related fitness items, flexibility, muscle strength and endurance, and cardiovascular functions. 80 subjects were students in Grades 1, 2, 3. Data were submitted to a step-wise multiple linear regression for each criterion variable. Predictor variables were age, sex, and body composition. Age was a significant factor in predicting performance on all variables except muscle strength, endurance, and flexibility. Sex significantly predicted performance for only flexibility and cardiovascular function and body composition for the power and cardiovascular function variables. Beyond the biological potential of each individual are factors that influence his motor development. These factors need early identification to make possible opportunities for each person to reach the full perimeters of motor potential.

Age Factors↗