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At least 1,531 records · Page 85Linked to original sources
PubMed2001

Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos.

The yield and quality of (a) parthenogenetic blastocysts produced by two activation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) and (b) nuclear transfer blastocysts generated using these two activation treatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optimal nuclear transfer protocol. The two activation protocols comprised calcium ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yields were greater (P < 0.001) following activation with DMAP than CHX (59.7 +/- 5.1 vs. 31.4 +/- 4.5 [mean +/- SEM]). In contrast, nuclear transfer blastocyst rates per fused embryo were lower (P < 0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7.3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst rate per fused embryo was negatively correlated (P = 0.0091) with the total number of blastomeres per donor embryo. Despite this inverse relationship, the calculated potential blastocyst yield per donor embryo was positively correlated (P < 0.0048) to karyoplast age. The individual potential yields on days 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4.7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relationship is that differentiation events during early cleavage are able to reduce the ability of the cytoplast to reprogram the transferred karyoplast and hence reduce blastocyst yields. The mechanism that mediates the differential effect of the CHX and DMAP on blastocysts yields between parthenogenetic and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer.

Animals↗
PubMed2004

Quantitative proton magnetic resonance spectroscopy of the cervical spinal cord.

Proton MR spectroscopy ((1)H-MRS) provides indices of neuronal damage in the central nervous system (CNS); however, it has not been extensively applied in the spinal cord. This work describes an optimized proton spectroscopy protocol for examination of the human cervical spinal cord. B(0) field mapping of the cord revealed periodic inhomogeneities due to susceptibility differences with surrounding tissue. By combining field maps and experimental data, we found that the optimum voxel size was 9 x 7 x 35 mm(3) placed with the inferior end of the voxel above vertebral body C2. Metabolite concentrations were determined in the cervical cord in six healthy controls by short-echo point-resolved spectroscopy (PRESS) volume localization. The results were compared with metabolite concentrations in the brainstem, cerebellum, and cortex in the same individuals. The concentrations in the cervical cord were as follows: N-acetyl-aspartate (NAA) 17.3 +/- 0.5, creatine (Cr) 9.5 +/- 0.9, and choline 2.7 +/- 0.5 mmol/l. The NAA concentration was significantly lower in the cord than in the brainstem (Mann-Whitney, P < 0.025), and higher than in the cortex (P < 0.005) and cerebellum (P < 0.005). Cr was significantly lower in the cord than in the cerebellum (P < 0.05). There were no significant differences between Cr concentrations in the spinal cord compared to the cortex and brainstem.

Adult↗
PubMed2004

Effect of load during electrical stimulation training in spinal cord injury.

Electrical stimulation training is known to alter skeletal muscle characteristics after a spinal cord injury, but the effect of load on optimizing the training protocol has not been fully investigated. This study investigated two electrical-stimulation training regimes with different loads on intramuscular parameters of the paralyzed lower limbs. Six paraplegic individuals with a spinal cord injury underwent electrical stimulation training (45 min daily for 3 days per week for 10 weeks). One leg was trained statically with load, and the contralateral leg was trained dynamically with minimal load. Isometric force assessed with 35-HZ stimuli increased significantly in both legs from baseline, with the static-trained leg also being significantly higher than the dynamic-trained leg. The vastus lateralis muscle of the statically trained leg showed a significant increase in type I fibers, fiber cross-sectional area, capillary-to-fiber ratio, and citrate synthase activity when compared to both baseline and the dynamically trained leg. Relative oxygenation of the vastus lateralis muscle as determined by near infrared spectroscopy was also significantly greater after static training. This study indicates that the load that is applied to paralyzed muscle during an electrical stimulation training program is an important factor in determining the amount of muscle adaptation that can be achieved.

Adult↗
PubMed2005

Renal and urinary proteomics: current applications and challenges.

During the past few years, proteomics has been extensively applied to various fields of medicine including nephrology. Current applications of renal and urinary proteomics are to better understand renal physiology, to explore the complexity of disease mechanisms, and to identify novel biomarkers and new therapeutic targets. This review provides some examples and perspectives of how proteomics can be applied to nephrology and how experimental data can be linked to physiology, functional significance and clinical applications. In some instances, proteomic analysis can be utilized to generate a new hypothesis from a set of candidates that are obtained from expression studies. The new hypothesis can then be addressed rapidly by conventional molecular biology methods, as demonstrated by identification of an altered renal elastin-elastase system in diabetic nephropathy and alterations in the renal kallikrein-kallistatin pathway in hypoxia-induced hypertension. The strengths and limitations of proteomics in renal research are summarized. Optimization of analytical protocols is required to overcome current limitations. Applications of proteomics to nephrology will then be more fruitful and successful.

Animals↗
PubMed1998

Mass spectrometric determination of the sites of O-glycan attachment with low picomolar sensitivity.

A sensitive protocol for unambiguously and positively identifying O-glycosylation sites in glycopeptides is described, based on beta-elimination of the glycan chain(s) using NH4OH. On glycan elimination, NH3 is incorporated into the amino acid residue(s) to which the glycan(s) had been attached, to yield a modified amino acid residue having a distinct mass. Electrospray ionization collision-induced dissociation tandem mass spectrometry allows the released, modified peptide to be sequenced and the site(s) of the modified amino acid residue(s) to be identified. The protocol has been optimized using a series of structurally related O-glycopeptides, and standard conditions are recommended for handling unknowns. We demonstrate that site determination can be achieved using as little as 1 pmol of starting material.

Amino Acid Sequence↗
PubMed1994

National Institutes of Health Consensus Development Conference Statement. Ovarian cancer: screening, treatment, and follow-up.

The National Institutes of Health Consensus Development Conference on Ovarian Cancer-Screening, Treatment, and Follow-up brought together epidemiologists; obstetrician/gynecologists; gynecologic, medical, and radiation oncologists; and the public to address the following questions: (1) What is the current status of screening and prevention in ovarian cancer? (2) What is the appropriate management of early-stage ovarian cancer? (3) What is the appropriate management of advanced epithelial ovarian cancer? (4) What is the appropriate follow-up after primary therapy? and (5) What are the directions for future research? The consensus panel concluded that there is no evidence available as yet that the current screening modalities of CA 125 and transvaginal ultrasonography can be effectively used for widespread screening to reduce mortality from ovarian cancer nor that their use will result in decreased rather than increased morbidity and mortality. They recommended that further prospective research be done to evaluate this very important issue. Women with stage IA grade 1 and most IB grade 1 ovarian cancer do not require postoperative adjuvant therapy. Many remaining stage I patients do require chemotherapy. Subsets of stage I must be fully defined and ideal treatment must be determined. Women with stages II, III, and IV epithelial ovarian cancer (other than low malignant potential tumors) should receive postoperative chemotherapy. Physicians should be encouraged to discuss clinical trial participation with women, and women should be encouraged to participate. All women should have access to accurate and complete information regarding ovarian cancer. Furthermore, there must be no barriers to women's access to qualified specialists, optimal therapy, and protocols. The full text of the consensus panel's statement follows.

Combined Modality Therapy↗
PubMedDate not supplied

Real space refinement of acto-myosin structures from sectioned muscle.

We have adapted a real space refinement protocol originally developed for high-resolution crystallographic analysis for use in fitting atomic models of actin filaments and myosin subfragment 1 (S1) to 3-D images of thin-sectioned, plastic-embedded whole muscle. The rationale for this effort is to obtain a refinement protocol that will optimize the fit of the model to the density obtained by electron microscopy and correct for poor geometry introduced during the manual fitting of a high-resolution atomic model into a lower resolution 3-D image. The starting atomic model consisted of a rigor acto-S1 model obtained by X-ray crystallography and helical reconstruction of electron micrographs. This model was rebuilt to fit 3-D images of rigor insect flight muscle at a resolution of 7 nm obtained by electron tomography and image averaging. Our highly constrained real space refinement resulted in modest improvements in the agreement of model and reconstruction but reduced the number of conflicting atomic contacts by 70% without loss of fit to the 3-D density. The methodology seems to be well suited to the derivation of stereochemically reasonable atomic models that are consistent with experimentally determined 3-D reconstructions computed from electron micrographs.

Actins↗
PubMed1998

An improved PCR-based method for site directed mutagenesis using megaprimers.

An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.

DNA Primers↗
PubMed2002

How to avoid spurious cluster validation? A methodological investigation on simulated and fMRI data.

This paper presents an evaluation of a common approach that has been considered as a promising option for exploratory fMRI data analyses. The approach includes two stages: creating from the data a sequence of partitions with increasing number of subsets (clustering) and selecting the one partition in this sequence that exhibits the clearest indications of an existing structure (cluster validation). In order to achieve that the selected partition is actually the best characterization of the data structure, previous studies were directed to find the most appropriate validity function(s). In our analysis protocol, we first optimize the sequence of partitions according to the given objective function. Our study showed that an insufficient optimization of the partition, for one or more numbers of clusters, can easily yield a spurious validation result which, in turn, may lead the analyst to a misleading interpretation of the fMRI experiment. However, a sufficient optimization, for each included number of clusters, provided the basis for a reliable, adequate characterization of the data Furthermore, it enabled an adequate evaluation of the validity functions. These findings were obtained independently for three clustering algorithms (representing the hard and fuzzy clustering variant) and three up-to-date cluster validity functions. The findings were derived from analyses of Gaussian clusters, simulated data sets that mimic typical fMRI response signals, andreal fMRI data. Based on our results we propose a number of options of how to configure improved clustering tools.

Algorithms↗
PubMed1997

Activation or frustration of anti-tumor responses by T-cell-based immune modulation.

Many types of tumors (e.g. virus-induced tumors, melanomas, tumors over-expressing certain oncogenes) often express antigens that can induce T-cell-mediated tumor-specific immune responses. Nonetheless, many such tumors manage to circumvent the induction of an effective anti-tumor T-cell response, as is apparent from the many tumor-bearing patients. Therefore, optimally designed vaccination protocols may evoke a more powerful and competent T-cell-mediated anti-tumor response, allowing the host to effectively deal with at least some cancers. These vaccination approaches might include immunization with whole (tumor) cell-based vaccines, entire tumor antigens or selected T-cell epitopes derived from tumor antigens. This survey is an update of several anti-tumor vaccination approaches employed, and on novel possibilities to target the anti-tumor immune response to preselected tumor-derived T-cell epitopes.

Adenoviridae↗
PubMed1993

Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes.

Conditions suitable for immunogold detection of digoxigenin-labelled DNA probes hybridized to parvovirus B19-infected erythroid cells embedded in Lowicryl K4M and LR White acrylic resins were established at the electron microscope level. The protocol was initially optimized using a positive control probe for whole human DNA which produced signal over the heterochromatin of all nucleated cells. In cultures harvested 2 days postinfection, B19 nucleic acid was detected mainly within the centrinuclear region of erythroid cells exhibiting characteristic margination of the chromatin. The B19 hybridization signal was largely unaffected by denaturation and was resistant to RNase digestion but sensitive to DNase digestion, indicating that it was mainly single-stranded B19 DNA. Relatively few gold particles were found over crystalline arrays of viral capsids, consistent with the observation that they are composed of mainly 'empty' capsids. B19 nucleic acid was detected in apparent transit from nucleus to cytoplasm through pores in the nuclear membrane. While the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section, it is a rapid and specific means of localizing viral nucleic acids with a high degree of resolution.

Cells, Cultured↗
PubMed1993

Postoperative PSK and OK-432 immunochemotherapy for patients with gastric cancer.

We evaluated the effects of chemotherapy given postoperatively with and without immunomodulators on the survival of patients who had undergone resection for gastric cancer. We conducted a retrospective survey of data on 963 Japanese patients treated at our department of surgery between 1965 and 1987. Data related to the duration of postoperative survival were calculated for those who received chemotherapy, i.e. an individualized combination of various agents given with or without the immunomodulators PSK, a protein extract of the fungus Coriolus versicolor, and/or OK-432, a preparation of an attenuated strain of Streptococcus (immunochemotherapy). Postoperative immunochemotherapy was more often prescribed for patients with advanced disease. The survival of patients who received immunochemotherapy was shorter than that of patients who received only chemotherapy. In a subgroup of patients adjusted for disease stage, the survival of those on chemotherapy versus immunochemotherapy did not differ significantly at any stage. For optimal results, a protocol for postoperative immunochemotherapy needs to be designed and investigated prospectively and according to the stage of gastric cancer. The stage III gastric cancers seem amenable to a favorable response.

Adjuvants, Immunologic↗
PubMedDate not supplied

Myocardial kinetics of radiolabeled perfusion agents: basis for perfusion imaging.

The myocardial deposition of radiolabeled perfusion agents permits the noninvasive assessment of regional coronary blood flow. The design of imaging protocols and the optimal interpretation of clinical perfusion studies are based on an understanding of the kinetics of blood-tissue exchange for these compounds. Thallium 201 and the technetium 99m-labeled compounds sestamibi, teboroxime, and tetrofosmin show differing myocardial extraction and retention. This review focuses on studies that used cell culture, isolated heart, and intact animal models that form the basis of our current understanding of the myocardial kinetics of these imaging agents.

Cells, Cultured↗
PubMed1997

Clinical utility of contrast-enhanced MR angiography.

MR angiography (MRA) is a technique under ongoing discussion. Its non-invasiveness and sensitivity to flow irregularities make it an investigational technique which is easy to apply but which does not always lead to comprehensive results. It requires special skill to perform and also experience for correct interpretation of the results. The lengthiness of the procedure combined with certain physical properties tends to limit its use to mostly neurovascular applications. With the introduction of ultrafast MRA in conjunction with peripheral bolus-injection of extracellular contrast media, a new approach to the imaging of such regions as the thoracic and abdominal vasculature not to mention other vascular territories has become possible. In this paper, considerations of bolus and measurement optimization and timing protocols for dedicated indications are presented together with an overview on the experience acquired to date for CE-MRA of the carotid artery, mediastinal and pulmonary vessels, abdominal vasculature, and peripheral vessels. The main advantage of ultrafast CE-MRA is that patients are subjected to much more tolerable breath-hold investigations with the result that physiological motion such as peristalsis or major pulsation is minimised.

Adolescent↗
PubMed1997

Indication-related dosing for magnetic resonance contrast media.

This presentation reviews the issue of contrast media dosing and imaging protocols for the optimal MR imaging detection and characterization of pathology. The cumulative clinical experience gained in performing contrast-enhanced MR examinations with gadolinium chelates indicates that a dose of 0.1 mmol/kg body weight provides safe and effective enhancement of most CNS pathology. Doses lower than 0.1 mmol/kg have been shown to be inadequate for delineating all but selected types of CNS pathology, such as masses with a high lesion to background ratio on post-contrast images (acoustic neuromas) or lesions located in areas in which the normal tissue very rapidly takes up contrast agent (e. g. microadenomas in the pituitary gland). Recent clinical studies have suggested a role for high dose gadolinium administration (up to 0.3 mmol/kg) for the optimal detection and delineation of cerebral metastases or other small or poorly enhancing lesions. Differences in the histopathologic characteristics (capillary permeability, vascularity, location, size) of specific diseased tissues may require varying doses or even a different contrast agent to be used for optimal imaging results. As new MR contrast agents and new scanning techniques are introduced, the specific diagnostic question posed will likely determine the choice of pulse sequence, contrast agent and dose used.

Contrast Media↗
PubMed1998

[Xenon-CT and perfusion MRI in the diagnosis of cerebral gliomas].

Within certain limits the vascularity of cerebral gliomas correlates with dysplasia. Xenon CT and perfusion MRI can be used to investigate tumor vascularity. Using an optimized wash in/out protocol with inhalation of 30% xenon gas, xenon CT was performed and regional cerebral blood flow (rCBF) and blood/tissue partition coefficient (lambda) calculated. Furthermore, perfusion MRI was performed using a dynamic T2*w EPI sequence with intravenous administration of Gd-DTPA. The relative regional cerebral blood volume, arrival time, time to peak and mean transit time were calculated. Both modalities were used in patients with supratentorial gliomas and compared with conventional contrast-enhanced MRI (ceMRI). Calculation of regional cerebral blood flow, as compared to ceMRI, did not contribute to the diagnostic approach of tumor dysplasia. Tumour vascularity was better estimated by measurement of lambda. Measurement of relative regional cerebral blood volume also correlated with tumor vascularity. Mainly the possibility of direct quantification of the Xe-CT data was advantagenous compared to perfusion MRI, and the XeCT data could be interpreted more clearly. Further upgrades of the technical equipment of MR as well as upgrades of available software will increase the applicability of perfusion MRI. However, both modalities offer a more precise investigation of tumor vascularization compared to ceMRI since potential dysplastic alteration may be detected earlier, which will be advantageous for planning bioptical approach of such tumours.

Astrocytoma↗
PubMed2003

PCR-multiplexes for a genome-wide framework of simple sequence repeat marker loci in cultivated sunflower.

Simple sequence repeat (SSR) and other DNA sequence-tagged site markers can be genotyped more rapidly and cost efficiently by simultaneously amplifying multiple loci (multiplex PCR). The development of PCR-multiplexes for a nearly genome-wide framework of 78 SSR marker loci in cultivated sunflower ( Helianthus annuus L.) is described herein. The most outstanding single-locus SSR markers in the public collection (300 out of 1,089) were identified and screened for polymorphisms among 24 elite inbred lines, preparatory to selecting SSR markers for testing in multiplex PCRs. The selected SSR markers produced robust PCR products, amplified a single locus each, were polymorphic among elite inbred lines (minimum, mean and maximum heterozygosities were 0.08, 0.53 and 0.85, respectively), and supply a dense genome-wide framework of predominantly or completely codominant, single-locus DNA markers for molecular breeding and genomics research in sunflower. Thirteen six-locus multiplex PCRs were developed for 78 SSR marker loci strategically positioned throughout the sunflower genome (three to five per linkage group) by identifying compatible SSR primer combinations and optimizing multiplex PCR protocols. The multiplexed SSR markers, when coupled with 17 complementary SSR marker loci, create a 'standard genotyping' set ideal for first-pass scans of the genome, as are often needed when screening bulked-segregant DNA samples or mapping phenotypic trait loci. The minimum, mean and maximum heterozygosities of the multiplexed SSR markers were 0.38, 0.62 and 0.83, respectively. The PCR-multiplexes increase genotyping throughput, reduce reagent costs, and are ideal for repetitive genotyping applications where common sets of SSR marker loci are required or advantageous.

Chromosome Mapping↗
PubMed2006

Molecular study of Mhc-DRB in wild chacma baboons reveals high variability and evidence for trans-species inheritance.

The MHC class II genes of many primate species were investigated extensively in recent years. However, while Mhc-DRB genes were studied in Old World monkeys such as rhesus macaques, the Mhc-DRB of baboons was only studied in a limited way. Because of their close anatomical and physiological relationship to humans, baboons are often used as models for reproduction and transplantation research. Baboons are also studied as a model species in behavioural ecology. Thus, identification of MHC genes would provide a foundation for studies of Mhc, biology and behaviour. Here, we describe the use of PCR, cloning, denaturing gradient gel electrophoresis (DGGE) and sequencing to identify Mhc-DRB sequences in wild chacma baboons (Papio ursinus). We amplified the highly variable second exon of baboon Mhc-DRB sequences using generic DRB primers. To validate and optimize the DGGE protocol, four DNA samples were initially studied using cloning and sequencing. Clones were screened using a novel RFLP approach to increase the number of clones identified for each individual. Results from cloning and sequencing were used to optimise DGGE conditions for Mhc-DRB genotyping of the remaining study subjects. Using these techniques, we identified 16 Paur-DRB sequences from 30 chacma baboons. On the basis of phylogenetic tree analyses, representatives of the Mhc-DRB1 and Mhc-DRB5 loci, and 13 different DRB lineages were identified. Evidence for trans-species inheritance of some Mhc-DRB sequences comes from high identity between the new Paur-DRB sequences and sequences from Papio cynocephalus, Macaca mulatta and possibly Galago moholi.

Amino Acid Sequence↗