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At least 163 records · Page 9Linked to original sources
PubMed1992

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

DNA Repair↗
PubMed2025

Systematic Optimization Enables Near-Perfect In Vitro Transformation Efficiencies for Spirodela polyrhiza (Greater Duckweed).

The in vitro transformation of plants, or the delivery of foreign genetic material that is incorporated into their genomes, represents a powerful tool both for elucidating genotype-phenotype relationships and for generating plant cultivars which have desirable traits for agriculture and/or biotechnological applications. However, outside of a few model species, the processes involved in transformation are often inefficient and can take months to perform for many plant species, with several bottlenecks occurring at the different stages of calli induction, genetic transfection, and plant regeneration. While duckweeds - aquatic monocots whose species include some of the smallest and fastest-growing flowering plants on the planet - have distinguished themselves with several emerging biotechnological applications, they too are the subject of conflicting reports regarding their transformation potential and ability to be genetically manipulated. Here, we synthesized and optimized the protocols for in vitro transformation of duckweed Spirodela polyrhiza (Greater Duckweed) from start-to-finish: achieving >90% - 100% efficiencies for each of calli induction; transient and stable genetic transformation; visual marker-free selection of transformants; and regeneration of genetically modified plants with stable transgene expression for over 100 generations - and which in S. polyrhiza can be achieved over the course of weeks instead of months. The integrated, streamlined approaches for all stages of in vitro transformation overcome many bottlenecks and can help to pave the way for high-throughput functional genomics studies and synthetic biology applications in this biotechnologically-important species.

CRISPR/Cas9↗
PubMed1990

Ligaments of the lateral aspect of the ankle and sinus tarsi: an MR imaging study.

The normal anatomy of the lateral ankle and subtalar ligaments seen at magnetic resonance (MR) imaging was studied in four cadaver ankles. Sixteen ankles of 11 healthy volunteers were imaged with four different MR imaging protocols to optimize technique. The anterior talofibular ligament was identified in 100% of the ankles of the volunteers in the axial plane, the calcaneofibular ligament in 81% of the ankles in the coronal plane, the cervical ligament in 69% of the ankles in the coronal plane and in 88% of the ankles in the sagittal plane, and the talocalcaneal ligament in 56% of the ankles in the coronal plane and in 62% of the ankles in the sagittal plane. It is concluded that thin-section (1-3-mm) MR imaging techniques, especially the one in which data are acquired with three-dimensional Fourier transform, are best for visualization of the ligaments. These techniques may play a role in the evaluation of patients with chronic ankle pain and instability.

Adult↗
PubMed1983

Lymphocytes bearing both T and B markers in lymphoproliferative disorders.

The appearance of dual-marked (D) lymphocytes, i.e. lymphocytes having both T and B markers, was investigated in patients with acute (ALL) and chronic lymphocytic leukaemia (CLL) as well as with non-Hodgkin's lymphoma (NHL). E-rosette and immunobead-rosette techniques were combined and simultaneously administered, and the combination was found to be simple, fast and reproducible. D cells could be detected also in normal peripheral blood, however, only in a low percentage. In the blood of 4 patients suffering from ALL, in 2 with CLL and in 1 with NHL the number of D cells were increased significantly. In ALL the relative number of these cells decreased during remission and increased in relapses; thus the determination of the relative frequency of D cells might have prognostic value and could be useful in the choice of an optimal therapeutic protocol.

Adult↗
PubMed1991

Oxytocin pharmacodynamics: effect of long infusions on uterine activity.

Despite common use in obstetrics for almost 50 years, there is still disagreement concerning optimal clinical protocols for the use of oxytocin. This disagreement arises in part from inadequate data on oxytocin pharmacokinetics and pharmacodynamics. This report evaluates the uterine response to fixed doses of oxytocin (1-3 mIU/min) over long infusion times (up to 120 min) in 10 patients with secondary arrest of dilatation. The uterine activity increased from 132 +/- 61 Alexandria units during the control period to 199 +/- 64 Alexandria units at an infusion rate of 1 mIU/min. Increasing the infusion rate to 2 mIU/min increased the uterine activity to 240 +/- 64 Alexandria units. The uterine activity increased until a steady state was achieved between 40 and 70 min after the initiation of infusion. Continuing the infusion at a fixed rate for more than 90 min appears to result in a decreasing uterine activity. Patients requiring oxytocin for augmentation of labor developed adequate uterine activity with dose rates of 1-3 mIU/min. Clinical protocols for oxytocin augmentation with an interval of 40-60 min between increases in the dosing seem reasonable based on these pharmacodynamic data.

Dose-Response Relationship, Drug↗
PubMedDate not supplied

Automated multiple peptide synthesis.

A fully automated instrument for multiple simultaneous peptide synthesis was constructed to provide large numbers of peptides for immunological research. The synthesis is performed in a flow-through mode with the conventional solid supports contained in 48 individual reaction columns. The instrument is based on a commercial autosampler equipped with a motor-driven syringe for accurate delivery of reagents and a robot arm carrying a dispenser needle. Dedicated software was developed to compile overlapping peptides from a given protein sequence and to control all functions of the robot. In situ activation by BOP was chosen as the optimized chemistry protocol. The peptides are cleaved from the resin in the reactors used for synthesis, thus minimizing handling. Performance of the instrument was demonstrated by synthesis of overlapping 14-mer peptides derived from the sequence of HIV reversed transcriptase. A second mode of operation allows the synthesis to be carried out on the surface of polyethylene pins. Peptides derived from the sequence of human TNF were synthesized using this method and used to characterize antibodies raised against the intact protein.

Amino Acid Sequence↗
PubMedDate not supplied

Effect of OK-432 on cytotoxic activity in cancer patients without tumor burden.

The present study was designed to determine the optimal therapeutic protocol for OK-432, a streptococcal preparation, and to clarify the kinetics of various immunological parameters following intracutaneous administration of OK-432 in 39 disease-free postoperative patients with early cancer. These patients were randomly divided into 4 groups according to the dose (1KE or 5KE) and the frequency of administration (one or 3 times every other day) of OK-432. NK activity 3-4 days after the first injection increased significantly in patients whose pretreatment baseline activity was below the lower level of normal values (60 LU) in all study groups (P < 0.05). The elevation of post-therapeutic NK activity lasted for 2 weeks in the group of patients given repeated injections. The serum content of cytotoxic activity against L-M cells increased significantly after the injection of OK-432 at doses of more than 5 KE doses (p < 0.05). These findings suggest that repeated injections of OK-432 over a period of a week have important immunotherapeutic results.

Adult↗
PubMed1992

Changes in actin state and chemotactic peptide receptor expression in granulocytes during cytokine administration after autologous bone marrow transplantation.

We studied the changes in actin state and chemotactic peptide receptor expression in granulocytes from patients receiving different cytokines following high dose chemotherapy and autologous bone marrow transplantation (ABMT). The F-actin content in granulocytes was higher in all patients following ABMT. However, in patients receiving granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF) the increase in F-actin content was much greater than in those not receiving these cytokines (159, 149, and 90% for G-CSF, M-CSF, and noncytokine group, respectively). Patients receiving granulocyte-macrophage colony-stimulating factor (GM-CSF) had only a 62% increase in the F-actin content, which was not statistically significant from patients undergoing ABMT without any cytokines. Although the basal level of F-actin was high following ABMT, granulocytes from all patients showed an additional increase in F-actin content after stimulation with either the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). The chemotactic peptide receptor expression was significantly higher in patients treated with ABMT alone or ABMT plus G-CSF. These observations suggest that the granulocytes generated following ABMT and cytokine administration may have different functional potential depending on the cytokine administered. Further studies to evaluate these potential differences are essential to devise optimal therapeutic protocols for maximizing the granulocyte protective function in this clinical setting.

Actins↗
PubMed1992

Computed tomographic evaluation of silicosis and coal workers' pneumoconiosis.

The introduction of computed tomography (CT) has modified the radiologic approach to silicosis and coal worker's pneumoconiosis considerably, allowing earlier recognition and more precise characterization of pulmonary abnormalities than chest radiography. An optimal approach to CT recognition requires an understanding of the main physiologic and pathologic features that are known to occur in both pneumoconioses. This report focuses on the most common CT features of simple and complicated forms of silicosis and coal worker's pneumoconiosis as well as on the optimal scanning protocol. The respective roles of chest radiographs and CT scans in the recognition of pneumoconiosis and follow-up of exposed workers are discussed.

Coal Mining↗
PubMed1991

Effect of dose on the pharmacokinetic and pharmacodynamic effects of cytarabine.

In summary, there are compelling laboratory and clinical data indicating that higher doses of ara-C than are currently used in SDaC protocols constitute optimal therapy. The cellular pharmacokinetics of ara-C are optimized at extracellular drug concentrations in the 10 to 15 mumol/L range. At these concentrations, transport rates are no longer rate-limiting, and ara-C phosphorylation capacity is saturated. The prime determinants of ara-C effect then shift to multiple intracellular events including anabolism to nucleotides, catabolism via deamination by Cyd-dCyd deaminase and dCMP deaminase, half-life of ara-CTP, the extent of incorporation into DNA, and the half-life of ara-CMP residues in DNA. It is postulated that at these high doses an additional effect of ara-C occurs on the cell membrane through affects on membrane phospholipid synthesis. This effect may contribute to the brisk cell lysis associated with HiDaC treatment. When administered as repetitive doses of 3 g/m2 over a 1- to 3-hour period, systemic deamination of ara-C gives rise to high plasma concentrations of ara-U. This metabolite has a long plasma half-life and, at least in the mouse, is concentrated in the liver and kidneys. High concentrations in these organs retard the further catabolism of ara-C and thus increase the systemic AUC providing a longer exposure period to the drug. A similar mechanism may obtain in patients treated with HiDaC. The observed decreased clearance of ara-C when administered in gram versus milligram doses and the long-terminal gamma-phase in plasma clearance of the drug associated with HiDaC usage quite probably reflects this effect of ara-U in patients. Additionally, by some as yet unknown mechanism, high concentrations of ara-U cause accumulation of leukemia cells in S-phase, the phase of the cell cycle wherein ara-C is maximally effective. This effect of ara-U may add to the cytokinetic effects initiated by rapid cytoreduction, which summate in the observed enhancement of the proliferative fraction of residual leukemia cells on day 8. The effect of a second course of therapy at this time is thereby enhanced. These dose-related and metabolite-drug interactions that occur when ara-C is given at high doses constitute a means for "self-potentiation" and may thus contribute to its overall therapeutic efficacy.

Animals↗
PubMed1991

Role of imaging in the evaluation of pelvic cancer.

The last 15 years have witnessed dramatic advances in the detection, assessment, and management of cancer. Progress is due to increased knowledge of the basic biology of cancer, oncogenes, and immunologic mechanisms controlling cancer; a better understanding of the disease and its diagnosis and management; and improved imaging technology. Diagnostic imaging has been revolutionized by the development of microcomputers and television instrumentation, and by technological advances producing better ultrasound transducers, x-ray detectors, and more homogeneous magnetic fields, as well as more precise magnetic coils and better software. At the time that we have come to understand more about cancer prognostic variables, imaging has developed to offer a unique display of a number of morphologic factors such as tumor size, location, depth of invasion, lymph node status, presence of metastases, and overall stage of disease. Just as accurate knowledge of prognostic variables is essential for designing optimal treatment protocols, it is fundamental in rendering a useful radiology report. Because "the first treatment should be the best possible since it has the greatest chance for curing the patient," an assessment of the full extent of cancer prior to the initiation of treatment is mandatory. Two elements in the clinical setting contribute to the ability to capitalize on these advances and breakthroughs: continuing education about progress in the field of oncology, and a motivated team of treatment specialists. This combination of application of new knowledge and technological advances and team work will promote the necessary environment for the conquest for cancer.

Female↗
PubMed1991

High-frequency ventilation: issues of strategy.

Although each high-frequency ventilator has functional characteristics that are design related, it now appears that when used with similar treatment strategies and within their functional limitations, similar clinical outcomes can be realized. Optimized treatment protocols must vary depending on the pulmonary disease to be treated, with strategies during treatment for pulmonary injury different from those used in pulmonary injury prevention. It is still unclear whether any one particular high-frequency ventilator will have specific disadvantages that cannot be overcome by device modification and that are inherent in a particular HFV methodology. Experimental and clinical data suggest that HFV will contribute significantly in reducing neonatal pulmonary morbidity and aid in the prevention of chronic pulmonary disease.

Clinical Protocols↗
PubMed1989

Therapy of dermatologic disease with cyclosporin A.

Cyclosporin A is a potent immunosuppressive drug with almost a decade of clinical use. Due to its lack of bone marrow toxicity, it has assumed a leading role in organ transplant surgery. Likewise, it has shown remarkable efficacy in psoriasis and has shown potential usefulness in other dermatologic diseases, most of which are thought to have an autoimmune T-cell-mediated pathogenesis. There are many other inflammatory skin conditions in which T lymphocytes represent a majority of the cellular infiltrates, and theoretically such disorders may be responsive to CsA. Diseases such as lichen planus, allergic contact dermatitis, photoallergy, and vitiligo are potential areas of further study. The potential use of topical and intralesional CsA also warrants further clinical evaluation. There are reports of topical CsA showing effectiveness for alopecia areata and nickel contact sensitivity. Griffiths et al. treated six psoriatic patients with 2% CsA ointment twice daily for 4 weeks. The response to placebo or CsA at the end of the study was similar in all patients. Notably, there was no measured systemic absorption and no change in blood pressure or renal function. We had similar unimpressive results in several patients treated with a 10% solution of CsA; minimal to no response was seen. Of great benefit would be an effective topical preparation without systemic absorption or toxicity. Future studies will need to carefully evaluate the vehicle used and the strength of topical CsA employed. The role of CsA in dermatologic disease still needs much investigation, both at the clinical and basic science levels. The studies to date show dramatic and exciting results. Indeed, CsA may well represent a major advance in the understanding and treatment of psoriasis. However, the decision to use CsA for nonfatal diseases such as psoriasis will require a careful balance between efficacy and toxicity. If long-term side effects can be avoided or kept within reasonably safe, acceptable limits, CsA holds great promise for the treatment of psoriasis. It may at times be used as an acute intervention, with a relatively large, initial dose for a short period of time to quickly clear severe psoriasis. This could be followed by either a reduction to a lower and safer maintenance dose or a program of other treatments. Cyclosporin A may also prove useful alone in very low doses or in combination therapy, as with UVB or PUVA. Optimal dosing protocols are currently under investigation.

Cyclosporins↗
PubMedDate not supplied

An in vitro model for human endothelial cell seeding of a small diameter vascular graft.

A precise system was devised to measure the kinetics of attachment of human venous endothelium to a variety of materials and substrates. Cells were labelled in a postconfluent state with tritiated thymidine, harvested, and a cell suspension seeded into a 4 mm PTFE graft. After a 90 minute incubation period, one half of the graft segment was sacrificed and the remaining portion placed in a perfusion system (225 cc/min) for 1 hour. Graft segments, effluents, and seeding suspension were assayed in a beta scintillation counter. The percentage of cells that attached pre- and postperfusion were determined, as well as the retrieval of tritium from the system. Initially, 71% of seeded cells attached to grafts coated with fibronectin, with significantly less (60%) remaining attached after perfusion. Only 10% of cells initially attached to uncoated grafts, with 4% retained postperfusion. Retrieval of tritium averaged 102 +/- 10% for all experiments. This system determines both pre- and postperfusion attachment of human endothelial cells to vascular grafts following manipulation of numerous variables, including graft material, substrate, incubation time, and seeding density. An optimal seeding protocol for human trials can thus be determined.

Blood Vessel Prosthesis↗
PubMed1986

General concepts and mechanisms of ventricular tachycardia.

The predominant mechanism of ventricular tachycardia is thought to be reentry, although this has been proved to exist in definitely a minority of patients studied owing to technical limitations. Other mechanisms, such as spontaneous triggered automaticity, undoubtedly also cause clinical ventricular tachycardia. During electrophysiologic testing, sustained unimorphic ventricular tachycardia can be induced in the majority of patients who have this arrhythmia clinically. It is extremely rare to induce this rhythm in a patient who has not had ventricular tachyarrhythmias. Nonsustained ventricular tachycardia, repetitive ventricular responses, and pleomorphic ventricular tachycardia and ventricular fibrillation are all nonspecific responses to electrical stimulation and thus are not suitable end points for either the baseline study or serial drug testing. Although an optimal pacing protocol for all subjects does not exist, an adequate test should include pacing with multiple drive rates, at least two (and in some cases, three) extrastimuli, and more than one pacing site in the right ventricle. Isoproterenol infusion and left ventricular pacing may also be necessary in selected patients. During serial drug testing, a drug should not be considered effective if more than 15 repetitive beats are induced unless an extremely aggressive pacing protocol is used. Patients treated with drugs predicted to be effective at electrophysiologic study have a better prognosis than those treated with drugs predicted to be ineffective or those treated with drugs selected empirically.

Electrophysiology↗
PubMed1982

[Current immunologic aspects of kidney transplantation].

The results of clinical kidney transplantation are mainly dependent on immunologic factors many of which are unknown or of unspecified importance. Blood transfusions have a favorable effect on graft prognosis, although our knowledge about optimal transfusion protocols and transfusion-induced mechanisms is still incomplete. The value of HLA-typing is controversial: whereas compatibility of the "classical" HLA-A,B,C antigens improves graft survival only moderately, HLA-DR typing, routinely performed for the last 3 years only, might be of greater importance. In addition, non-HLA systems, such as endothelial/monocytic antigens or the Lewis blood group system, appear to play a role in graft rejection. The individual immune reactivity a recipient to a large extent determines the fate of a graft. The multifactorial dependence of graft prognosis is discussed in this report.

Blood Group Incompatibility↗
PubMed1976

Randomized clinical trial of antithymocyte globulin in cadaver renal allograft recipients: importance of T cell monitoring.

Despite incontrovertable evidence demonstrating the unique immunosuppressive capabilities of antihymocyte globulin (ATG) in animals, its value in clinical transplantation has remained inconclusive. A multicenter, prospective study was undertaken in an attempt to determine this value. Cadaver renal allograft recipients were randomized into two treatment groups: prednisone and azathioprine only, or prednisone, azathioprine, and ATG. Four to 36 month follow-up of our first 50 recipients, supported by results in over 200 patients from other centers, permits the following observations. (1) Allograft survival can be improved by nearly 20 percent in recipients treated with active ATG preparations. (2) Presently available assays performed in vitro and on subhuman primate allograft survival, though capable of excluding inactive preparations, cannot quantitate precisely the immunosuppressive capacity of active batches of ATG. (3) Monitoring circulating T cell levels in patients receiving ATG appears to offer the best means of defining immunosuppressive potency and the variation in response to the agent among identically treated individuals. It is concluded that ATG therapy can be beneficial in renal transplantation; but monitoring of recipients' T cell level is desirable during therapy to determine the dosage required. Furthermore, the optimal immunosuppressive protocol can be approximated by modifying the regimen according to individual variations in response, thus limiting the infectious complications of excessive immunosuppression.

Adolescent↗