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Results for “Step selection function”

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PubMed1992

Rapid large-scale preparation of recombinant Erwinia chrysanthemi L-asparaginase.

L-asparaginase from Erwinia provides an alternative to the enzyme from E. coli for the effective treatment of acute lymphoblastic leukaemia. A procedure was required for the large-scale partial purification of the recombinant Erwinia enzyme cloned and expressed in Erwinia. Enzyme was extracted from Erwinia at high pH and extraneous protein precipitated at low pH. S-Sepharose FF was selected as the medium of choice for the chromatography step since it was adequate for the high flow rates required (linear flow rate 315 cm h-1) and the methylsulphonate functional groups exploited the high pI of the enzyme by allowing binding of L-asparaginase at pH 4.8 while most of the other proteins passed through the column. The useful capacity of the matrix was up to 34 mg enzyme/ml matrix at a linear flow rate of 95 cm h-1 and 15.4 mg enzyme/ml matrix at a linear flow rate of 315 cm h-1. Weakly bound protein was removed by a wash at pH 6.0. The L-asparaginase was eluted by a wash at pH 6.8 (linear flow rate 95 cm h-1) and was substantially pure, only requiring polishing steps to be suitable for use as a parenteral agent. The purity of the protein was complemented by a 92% recovery of active enzyme from this cation-exchange matrix.

Asparaginase↗

PubMedDate not supplied

Regioisomeric products of propranolol metabolism. The monomethyl ethers of 3,4-dihydroxypropranolol and of 3,4-dihydroxypropranolol glycol.

Regioisomeric monomethyl ethers of the 3,4-catechol of propranolol (1) and its 3-aryloxypropane-1,2-diol (glycol) metabolite were prepared to prove the structures of these putative products of oxidative metabolism. The ring regioisomer 4-methoxy-3-hydroxypropranolol (3) was prepared from 1-acetoxy-3-acetyl-4-methoxynaphthalene (8). Baeyer-Villiger oxidation was the key step in converting the 3-acetyl functionality to the desired 3-naphthol. The ring regioisomer 4-hydroxy-3-methoxypropranolol (4) was prepared from 3-methoxy-1,4-dihydroxynaphthalene (13) by selective 1-O-acylation with trimethylacetyl chloride. 4-O-Benzylation, followed by hydrolysis, and side chain elaboration afforded the 4-O-benzyl ether of 4. Similar methods afforded glycols 5 and 6, with the side chain obtained by osmium tetroxide oxidation of an O-allyl group. GC/MS analysis using the trifluoroacetyl derivatives of these known standards showed both 3 and 4 were metabolites of 1 in the rat. From a single dose study in man, 4 was identified as a minor urinary metabolite, and both regioisomeric glycol metabolites 5 and 6 were observed. In addition, another regioisomeric hydroxymethoxyglycol metabolite was found.

Animals↗

PubMedDate not supplied

Selection for surgery and long-term results in renovascular hypertension.

From 1960 to December 1977, 251 patients with renovascular hypertension (RVH) were observed; 219 were operated upon. Long-term results were assessed in 130 patients. Selection for surgery follows three steps: 1) screening of patients with renal artery disease, which is done by angiography; 2) diagnosis of RVH, which is reached mainly by differential renal function studies and renal vein renin measurements (ancillary methods are intravenous pyelography and sequential scintigraphy; the significance of renin measurements is discussed); and 3) prediction of the results of surgery and choice of technique. An original dynamic test of the authors is employed and reconstruction of the renal artery is the procedure of choice. This is done in the majority of cases by aortorenal bypass grafts using dacron prostheses. Techniques and complications are discussed and comparison with venous autograft is made. Hospital mortality was 3.2%. Overall long-term results were favorable in 78%. Long-term mortality was 6% and occurred mainly in patients who remained hypertensive. Results in atherosclerotic patients are compared with those obtained in fibrous stenoses. Results of renal artery reconstructions were far better than those of nephrectomies and lead us to restrict indications for such a procedure.

Aorta, Abdominal↗

PubMed1988

Selection of antibody repertoires by anti-idiotypes can occur at multiple steps of B cell differentiation.

These experiments were designed to evaluate whether alterations in Id expression after anti-Id treatments result from direct modulation of Id-producing B cells, and whether idiotypic selection operates in bone marrow or spleen B cells. By using the NPb Id model, we have studied the functional behavior of isolated LPS-reactive B cells transferred from B6 mice into histocompatible LPS-NR B10.Cr hosts and primed with LPS conjugates of anti-Id antibodies. We have found that previous anti-idiotypic manipulation of host mice by neonatal administration of suppressive doses of Ac 38 antibodies, or adult injection of enhancing doses of Ac 146 antibodies, modulated the T cell-independent Id response of either immature bone marrow or mature splenic responding cells, transferred from normal, untreated donors. These results are interpreted to suggest that selection of antibody repertoires by anti-Id may occur at multiple steps of B cell differentiation.

Animals↗

PubMed1986

Functional interdependence of discrete vagal projections to SA and AV nodes.

Dynamic modulation of chronotropic and dromotropic function was evaluated in anesthetized dogs before and after selective parasympathectomy of the atrioventricular (AV) node. Vagal and cardiac sympathetic efferent nerves were decentralized, and the distal cut ends of the cervical vagi were stimulated at frequencies from 1 to 25 Hz with step-wise voltage changes. To investigate parasympathetic modulation of AV conduction, stimulations were performed with and without atrial pacing. After determination of chronotropic and dromotropic responses, in the intact state, selective AV node parasympathectomy was performed as previously described [Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H61-H68, 1985]. Stimulation protocols were then repeated. Control results in intact animals reveal a parallel and finely balanced vagal regulation of chronotropic and dromotropic responses over a wide range of frequency stimulations. Conversely, selective parasympathectomy of the AV node interrupts extrinsic vagal modulation of AV conduction without affecting parasympathetic control of chronotropic function. The dynamic balance of chronotropic and dromotropic function, modulated by extrinsic vagal input, reflects the parallel activation of functionally and anatomically distinct parasympathetic projections to the sinoatrial (SA) and AV nodal tissue. Thus present experiments allow a more detailed examination of vagal innervation of the SA and AV nodes, the importance of their balance through neuroregulation, and progress toward understanding the pathophysiology of disturbances to this balance.

Animals↗

PubMed1986

Chromosomal localization of integrated adenovirus DNA in productively infected and in transformed mammalian cells.

The in situ hybridization technique has been used to localize adenovirus type 12 (Ad12) genomes on specific chromosomes of Ad12-transformed hamster cells as well as on specific chromosomes of human cells productively infected with Ad12. Hamster cell lines T637 and A2497-2 contain 20 to 22 and 17 copies of Ad12 DNA, respectively, in an integrated form. The results of in situ hybridization experiments demonstrate that the Ad12 DNA molecules are predominantly located on chromosomes 2, 7, 11 to 13, and 15 in cell line T637, and on chromosomes 7, 20, and perhaps 16 to 19 in cell line A2497-2. These data further document that viral DNA is chromosomally integrated and does not persist in a huge extrachromosomal, episomal structure. There is evidence from previous work that adenovirus DNA can also be covalently linked to human cellular DNA after productive adenovirus infection of human cells. In keeping with these earlier findings, we have now been able to show by in situ hybridization experiments that early in productive infection, i.e., 2 or 6 hr after infection, Ad12 genomes are predominantly associated with a limited number of human chromosomes. It appears biologically significant that these chromosomes, which belong to groups A and CII, have been the same ones in several independent infection experiments. Other chromosomes may also carry smaller amounts of viral DNA. In situ hybridization of Ad12 DNA to chromosomes of uninfected human cells yields no significant chromosomal association of viral DNA. The signal to noise ratio of grain counts over chromosomes from Ad12-infected cells to areas devoid of chromosomes is 5.6. The results presented raise the question of whether Ad12 DNA can integrate at selective sites in human chromosomes and whether this insertion event plays an essential role in early steps of viral transcription or replication. There is no direct evidence to suggest a biologic function for viral DNA integration in the productive infection cycle.

Adenoviruses, Human↗

PubMed1992

Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events.

WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

An independent application accuracy evaluation of stereotactic frame systems.

The purpose of incorporating stereotactic methodology into neurosurgical procedures is to consistently achieve a high degree of accuracy and precision in localizing intracranial targets. Therefore, the limits of resolution for the therapeutic intervention itself are a function of the accuracy and precision inherent to the particular stereotactic frame system itself. The total clinically relevant error (application accuracy) comprises errors associated with each procedural step, including imaging, target selection, vector calculation and the mechanical errors of stereotactic frames. To evaluate these parameters, a systematic error analysis was carried out in the 4 most commonly used CT-compatible stereotactic devices: the Brown-Roberts-Wells, Cosman-Roberts-Wells, Kelly-Goerss Compass (modified Todd-Wells) and Leksell frames. Over 7,681 independent test measurements were made. The results suggest a potentially significant degree of error in application accuracy of all stereotactic instrumentation which is accentuated by imaging-associated error. These individual error values must be considered with every clinical use of stereotactic frames.

Humans↗

PubMedDate not supplied

[Operative and diagnostic set-up for dysfunctional patients].

The application of the set-up for treatment planning and prognosis can be extended to the stimulation of the orthodontic teeth movements in the patients with TMJ dysfunctions, already repositioned, and with very severe dentoalveolar discrepancy in order to decide the indication for extractions and selection of the teeth to be extracted. The limits of this simulation always remain the function of the masticatory system. The sequences of the procedures on the articulator and the different steps on the upper and lower models are described. A patient with TMJ disorders and dento-alveolar discrepancy is presented with the different procedures of treatment as for the dysfunction as for the orthodontic problems.

Adult↗

PubMed1978

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids".

A plasmid which contains a cos site of lambda and can be packaged into lambda bacteriophage particles is termed a "cosmid". Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA. Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli "gene bank" was obtained with an efficiency of 5 . 10(5) clones per microgram of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones. It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.

Coliphages↗

PubMed1975

Genetic drift in clines which are maintained by migration and natural selection.

Genetic drift will cause a migration-selection cline to wobble about its expected position. A rough linear approximation is developed, valid when local populations are large. This is used to calculate effects of genetic drift on clines in a stepping-stone model with abrupt and with gradual changes of selection coefficients at a single haploid locus. Among the quantities calculated are measures of slope, standardized variation of gene frequencies around their expected values, and correlation among neighboring populations with respect to deviations from the expected gene frequencies. These quantities appear to be primarily functions of Ns and Nm for a given pattern of selection. Computer simulation gives rough confirmation of these results. Standardized variances of gene frequencies and correlation of neighbors differ along the cline in the case of smooth changes in selection. In no case is pathological behavior of gene frequency deviations found near the boundaries of selective regions. Local behavior of gene frequences of nearby colonies is approximately predicted by a simple adaptation of the stepping-stone theory of Kimura and Weiss. Approximate measures of the lateral variation of the midpoint of a cline and the probability of non-monotonicity are also calculated and discussed.

Gene Frequency↗

PubMed1992

Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain.

Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen.

Animals↗

PubMed1985

Stepwise variable selection and maximum likelihood estimation of smoothing factors of kernel functions for nonparametric discriminant functions evaluated by different criteria.

Kernel functions are used to estimate the probability density functions of variables for nonparametric discriminant analysis. In connection with stepwise variable identification a stepwise maximum likelihood estimation procedure for the estimation of smoothing factors of the kernel functions is developed. This procedure allows a step-by-step estimation of smoothing factors for every variable which is considered to be added to the model or which is examined to substitute a variable in a model. Different criteria for model evaluation in stepwise discriminant analysis are discussed. Beside criteria, like distance and dependence functions and the error and nonerror rate, a criterion which considers the ratio of probability densities of different classes at point x is proposed for stepwise variable identification. An application of the procedures described in this study to a medical decision problem shows the importance of stepwise parameter estimation of kernel functions for nonparametric discriminant analysis and the role of different model evaluation criteria for the selection of the best subset of variables.

Biometry↗

PubMed1992

Impaired cardiopulmonary response to exercise in moderate hypertension.

OBJECTIVE: To identify the limiting factors of exercise performance in subjects with hypertension associated with left ventricular hypertrophy. The secondary objective was to establish relationship between peripheral function and exercise capacity. DESIGN: Cardiopulmonary exercise testing was conducted using two protocols: a graded exercise test to maximal effort established maximal exercise capacity, followed by a step-incremental test combining gas-exchange measures and radionuclide angiography. The exercise responses were compared within and between groups. SETTING: All hypertensive subjects were selected from the Toronto Tri-Hospital Hypertension Clinic. Normal subjects were recruited from the surrounding community. PATIENTS: Twelve patients with established hypertension and left ventricular hypertrophy (determined by echocardiography) were studied as a referred/volunteer sample. All had no evidence of coincident diseases and were unmedicated at time of testing. A volunteer sample of normal, healthy subjects acted as a control. INTERVENTIONS: Graded exercise to maximum and step-incremental (submaximal and steady-state) exercise was used to quantify cardiopulmonary function during exercise stress. MAIN OUTCOME MEASURES: These included (for exercise performance) maximal oxygen intake (VO2max), the ventilatory anaerobic threshold, total peripheral resistance and blood lactate. Cardiac function measures included ejection fraction and ventricular volumes. RESULTS: Cardiac function data obtained during exercise in hypertensive subjects included an increase in the pressure to volume ratio, but a blunted ejection fraction response at peak exercise (P less than 0.05). Although end-diastolic volume increased during exercise (P less than 0.05), values were lower during both levels of exercise compared with normal subjects. Mean +/- SD end-systolic volume increased from 39 +/- 22 at rest to 42 +/- 23 mL during peak exercise. Hypertensive subjects had a lower VO2 max (mean 27.4 +/- 4.8 mL/kg/min) compared with normals (40.0 +/- 8.5 mL/kg/min) and a lower ventilatory anaerobic threshold (14.4 +/- 2.9 versus 27.6 +/- 5.8 mL/kg/min, P less than 0.005). Furthermore, hypertensive patients had a significantly elevated total peripheral resistance at rest (2.5 +/- 1.0 versus 1.8 +/- 0.4 peripheral resistance units) and at peak exercise (1.6 +/- 0.7 versus 0.8 +/- 0.2, P less than 0.01) compared with normal subjects (P less than 0.05). A correlation coefficient of 0.92 was found between total peripheral resistance and VO2 max in hypertensive subjects (P less than 0.01). CONCLUSIONS: These data suggest that peripheral factors, specifically a failure to reduce significantly total peripheral resistance, limits exercise performance despite a maintenance of left ventricular function during exercise in patients with moderate hypertension. The use of cardiopulmonary exercise testing can help in identifying the underlying cause of exercise intolerance in this population and limited left ventricular reserve at peak exercise, and may offer a sensitive measure of therapeutic end-points.

Adult↗

PubMed2024

Lineage-specific splicing regulation of MAPT gene in the primate brain.

Divergence of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is widespread in mammals, including primates, but the underlying mechanisms and functional impact are poorly understood. Here, we modeled cassette exon inclusion in primate brains as a quantitative trait and identified 1,170 (∼3%) exons with lineage-specific splicing shifts under stabilizing selection. Among them, microtubule-associated protein tau (MAPT) exons 2 and 10 underwent anticorrelated, two-step evolutionary shifts in the catarrhine and hominoid lineages, leading to their present inclusion levels in humans. The developmental-stage-specific divergence of exon 10 splicing, whose dysregulation can cause frontotemporal lobar degeneration (FTLD), is mediated by divergent distal intronic MBNL-binding sites. Competitive binding of these sites by CRISPR-dCas13d/gRNAs effectively reduces exon 10 inclusion, potentially providing a therapeutically compatible approach to modulate tau isoform expression. Our data suggest adaptation of MAPT function and, more generally, a role for AS in the evolutionary expansion of the primate brain.

tau Proteins↗

PubMed1991

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Human DNA can be cloned as yeast artificial chromosomes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.

Animals↗

PubMed1992

Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs.

In the past two decades many studies have been devoted to the involvement of the periportal (zone-1) and perivenous (zone-3) hepatocytes in bile formation and hepatobiliary transport of endogenous and exogenous compounds. It became clear that such a heterogeneity in transport function can, in principle, be due to the different localization of the cells in the acinus with respect to the incoming blood, to intrinsic differences between the cells or to both. In this review we first discuss the techniques used to study hepatocyte heterogeneity in hepatobiliary transport function. Combinations of such techniques can be used to discriminate between cellular heterogeneity due to acinar localization as opposed to intrinsic differences. These techniques include: normal and retrograde perfusions of isolated perfused livers; autoradiographic, fluorimetric and histochemical localization of injected substrates; separation of isolated hepatocytes into fractions enriched in periportal and perivenous cells; measurements of fluorescent surface signals with microlight guides; selective zonal toxicity, and pharmacokinetic modelling and analysis. Subsequently, for each of the rate-limiting steps in the hepatobiliary transport of organic compounds, the basic mechanisms are summarized and the available knowledge on the involvement of the cells from the various zones in these transport steps is discussed. The available literature data indicate that heterogeneity in transport function is often due to the localization of the cells in the acinus: the periportal cells are the first to come into contact with the portal blood and are thus exposed to the highest substrate concentration. Consequently they obtain the most prominent task in further disposition of the particular compound. It follows that the extent of involvement of the perivenous cells in drug disposition is implicitly determined by the activity of the periportal cells. Because of the potential saturation of elimination processes in the periportal cells, the involvement of perivenous cells may vary with the input concentration. In addition, real intrinsic differences have been established in the hepatobiliary transport of some substrates. These are probably based on differences in the cellular content of carrier- and receptor-binding and/or metabolizing proteins. In some cases these intrinsic differences may be secondary to existing sinusoidal gradients of endogenous compounds, such as O2, amino acids, bile acids or monosaccharides. Yet, data on the heterogeneity of hepatocytes in the various transport steps are far from complete or are even totally lacking, especially for human liver. A multi-experimental approach and advanced technology will be needed in the future to gain more insight into the acinar organization of bile formation and hepatobiliary transport of drugs in the human.

Animals↗

PubMed1989

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex.

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Animals↗