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At least 991 records · Page 55Linked to original sources
PubMed1994

Fluorescent differential display: arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.

We established robust, reliable protocols for 'Differential Display (DD),' an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967-971] using RT-PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such 'Fluorescent Differential Display (FDD)' techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.

Base Sequence↗
PubMed1995

Immunization against a rat colon carcinoma by sodium butyrate-treated cells but not by interleukin 2-secreting cells.

BACKGROUND & AIMS: Vaccination of patients with colon cancer with irradiated autologous tumor cells and bacille Calmette-Guérin (BCG) was reported to augment mean survival. It was recently observed that a local treatment combining recombinant interleukin 2 and the differentiation agent sodium butyrate cured rats with colon cancer peritoneal carcinomatosis. To optimize vaccination protocols, the comparison of the efficacy of irradiated tumor cells mixed with BCG with that of interleukin 2-gene-transfected cells and of tumor cells pretreated with sodium butyrate was performed. METHODS: The poorly immunogenic rat colon carcinoma cells PROb were used in a vaccination assay. Interleukin 2-transfected PROb cells, either proliferating or irradiated, were used. The efficiency of irradiated PROb cells mixed with BCG, of interleukin 2-transfected cells, or of cells pretreated with sodium butyrate was tested. RESULTS: Vaccination with irradiated parental cells and BCG did not provide protection. Irradiated interleukin 2-transfected cells were poorly efficient in the vaccination assay. Conversely, vaccination with irradiated parental cells pretreated with sodium butyrate before injection provided good protection. CONCLUSIONS: Interleukin 2-secreting cells efficiently vaccinated animals when injected while replicating but not after irradiation. Conversely, sodium butyrate pretreatment provided a simple and efficient vaccination scheme that generated a long-term immune memory and allowed the use of irradiated cells.

Animals↗
PubMed1990

The use of E. coli exonuclease III to generate single stranded DNA in BrdUrd cell-cycle analysis permits simultaneous detection of cell surface antigens.

An immunocytochemical method for the simultaneous flow cytometric quantitation of total cellular DNA, incorporated 5-bromo-2'-deoxyuridine (BrdUrd) and one or more cell surface antigens has been developed. Biotin labeling of cell surface antigens, critically tuned fixation techniques and an enzymatic denaturation of cellular DNA are the essential features of this method. Enzymatic denaturation of cellular DNA was shown to prevent loss of cell surface antigen-bound biotin moieties, and thus to preserve cell surface immunofluorescence distribution. After a mild protein extraction and the introduction of breaks into the chromatin using restriction endonucleases, E. coli exonuclease III was used to generate stretches of single stranded DNA. This approach permits detection of the incorporated BrdUrd using anti-BrdUrd monoclonal antibodies. The enzymatic denaturation protocol was optimized using in vitro BrdUrd-labeled L1210 murine leukemia cells, and applied to both in vivo and ex vivo BrdUrd-labeled murine bone marrow cells. With this new method it is possible to study DNA content, cell cycle kinetics and cell surface antigen expression simultaneously, and hence functional relationships between these parameters can be investigated.

Animals↗
PubMed1993

Conformation-sensitive immunoassay: optimisation, validation and evaluation.

A conformation-sensitive immunoassay (CSI) has recently been developed (Pfund and Bourdage, 1990). This paper describes the optimal laboratory protocol which makes this method useful as an early screening procedure for conformation-sensitive monoclonal antibodies. The method was validated with a panel of 12 monoclonal antibodies. Results from our experiments confirm that this variant of CSI can be applied in the analysis of purified proteins and glycoproteins, as well as complex mixtures of antigens.

Antibodies, Monoclonal↗
PubMed1987

Radioimmunoassay of epitestosterone: methodology, thermodynamic aspects and applications.

A radioimmunoassay system for 17 alpha-hydroxy-4-androstene-3-one (epitestosterone) was developed and evaluated using rabbit antisera against 17 alpha-hydroxy-4-androstene-3-one-3-(O-carboxymethyl)-oxime-bovine serum albumin conjugate, and radioiodinated homologous histaminyl derivative of epitestosterone as a tracer. Two antisera with different specificity were used for radioimmunological determination of epitestosterone in hydrolyzed urine and plasma, respectively. Apparent intrinsic association constants and derived thermodynamic variables for ligand-antibody interaction were measured under various conditions in order to set up an optimal assay protocol. The mean plasma epitestosterone levels for healthy subjects were 0.44 +/- 0.15 nmol.l-1 (means +/- SD) in males and 0.092 +/- 0.056 nmol.l-1 in females. The corresponding concentrations in urine were 320 +/- 70 nmol.l-1 (males) and 273 +/- 100 nmol.l-1 (females).

Body Fluids↗
PubMed1993

Isolation of a novel 45 kDa plasminogen receptor from human endothelial cells.

We have previously identified an endothelial cell membrane protein of M(r) 45 kDa that binds plasminogen in a kringle-dependent, specific and reversible manner (Dudani et. al. (1991) Mol. Cell. Biochem. 108: 133-139). In this study, we have developed and optimized a protocol for the isolation of the 45 kDa plasminogen receptor from venous endothelial cells using a four step procedure consisting of lysis and detergent extraction followed by ligand affinity chromatography and preparative polyacrylamide gel electrophoresis. Control experiments were carried out using BSA-Sepharose instead of plasminogen-Sepharose as the affinity matrix. No plasminogen binding proteins were recovered from the former columns. However, a 45 kDa protein was recovered from lysine eluates of plasminogen-Sepharose. This material was then purified to homogeneity using preoperative electrophoresis. Analyses of proteins at various steps in the purification by SDS-PAGE showed enrichment of a band of 45 kDa which superimposed with the observed binding activity of plasminogen in ligand blots. The above binding could be inhibited by excess lysine. The 45 kDa protein could be distinguished from alpha-enolase which also binds plasminogen by: (i) significant differences in the profile of retention times of CNBr-degradation fragments on reversed phase HPLC; and (ii) partial peptide sequencing of one of the CNBr-degradation fragments of the 45 kDa protein. Moreover, the derived sequence did not show any significant homology to any protein in the Swiss Prot (release 20) database. We thus propose that the 45 kDa protein represents a novel plasminogen receptor on human venous endothelial cells.

Amino Acid Sequence↗
PubMed1990

Aggressive chemosurgical debulking in patients with advanced ovarian cancer.

From July 1986 to June 1989, 43 evaluable patients with advanced ovarian cancer were treated on protocol with initial cytoreductive surgery, two courses of high-intensity intravenous Cytoxan (1000 mg/m2) and cisplatin (120-200 mg/m2) chemotherapy, and repeat debulking laparotomy in an effort to maximize response to a subsequent four cycles of intraperitoneal platinum-based chemotherapy. Two patients were stage IIIA, 2 stage IIIB, 28 stage IIIC, and 11 stage IV. Five tumors were grade 1, 9 grade 2, and 29 grade 3. Thirty-eight (88%) patients had bulky tumor (5-25 cm) found at first laparotomy; 25 of these had greater than 1-cm residual after initial debulking. Following two cycles of intensive intravenous chemotherapy 18 of these 25 had greater than 1-cm disease found at second laparotomy; 12 of 18 underwent secondary cytoreduction to less than 1 cm. Thus, 30 of these 38 (79%) patients entered the intraperitoneal phase of the protocol with less than 1-cm disease. Four patients had 2- to 5-cm tumor at initial laparotomy; two of four were debulked to less than 1-cm residual. All four were found to have less than 1-cm disease at second laparotomy. This combination regimen was well tolerated. There was one treatment-related death. In sum, 42 of 43 patients had tumor greater than 2 cm at staging laparotomy and 38 (88%) had large, bulky disease (5-25 cm); 34 of 43 (79%) entered the intraperitoneal phase of the protocol with optimal (less than 1-cm) disease. Aggressive chemosurgical cytoreduction in patients with bulky advanced ovarian cancer can leave a large proportion of patients with minimal residual disease and maximize their chances of responding to subsequent intraperitoneal chemotherapy.

Cisplatin↗
PubMed1990

Preparation of diethylaminoethyl hollow fibres for high-flow liquid chromatography.

A novel anion-exchange column for fast-flow liquid chromatography was developed. The column was prepared by immobilizing diethylaminoethyl groups on to the inner walls of regenerated cellulose hollow fibres. A detailed investigation was conducted of the parameters affecting diethylaminoethyl immobilization such as the NaOH concentration, the reaction temperature, the diethylaminoethyl concentration and the reaction span. Based upon the findings, a protocol to optimize the preparation of the diethylaminoethyl hollow fibres was established. The diethylaminoethyl hollow fibres prepared adsorbed 186 +/- 30 mg of albumin/g fibres.

Adsorption↗
PubMed1994

Postmortem anatomy from cryosectioned whole human brain.

A system of histologic and digital processing protocols are presented for the acquisition of high-resolution digital imagery from postmortem cryosectioned whole human brain and head for computer-based 3-dimensional (3D) representation and visualization. We designed and evaluated several protocols for optimal preparation of frozen specimens including fixation, decalcification, cryoprotection, freezing and sectioning procedures. High-resolution (1024(2) pixel) serial images were captured directly from the cryoplaned blockface using an integrated color digital camera and fiber optic illumination system mounted over a modified cryomacrotome. Specimens frozen and sectioned with the cranium intact preserved brain spatial relationships and anatomic bony landmarks. Color preservation was superior in unfixed tissue heads were incompatible with decalcification and cryoprotection procedures and section collection from such specimens was complicated by bone fragmentation. Collection of 1024(2) images from whole brain resulted in a spatial resolution of 200 microns/pixel in a 1-3 Gbyte data space. Even higher 3D spatial resolution was possible by primary image capture of selected regions such as hippocampus or brain stem. Discrete registration errors were corrected using image processing strategies such as cross-correlative and other algorithmic approaches. Data sets were amenable to resampling in multiple planes as well as scaling and transpositioning into standard coordinate systems. These methods enable quantitative measurements for comparison between subjects and to published atlas data. These techniques allow visualization and measurement at resolutions far higher than those available through other imaging technologies and provide greatly enhanced contrast for delineation of neuroanatomic structures, pathways, and subregions.

Brain↗
PubMed1991

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments.

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multi-site attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab') fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.

Adipates↗
PubMed1992

Chemiluminescent DNA probes: evaluation and usefulness in forensic cases.

Five phosphatase-labelled oligonucleotide probes were evaluated in respect to their sensitivity, with the help of an optimized chemiluminescent protocol, for DNA-VNTR polymorphism determination. Their usefulness for the identification of biological traces is illustrated with casework examples.

Alkaline Phosphatase↗
PubMed1992

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.

Base Sequence↗
PubMedDate not supplied

MRA-guided stereotactic radiation treatment planning for cerebral angiomas.

Stereotactic neurosurgical interventions are characterized by a high risk of rupturing intracranial vessels or damaging vital brain structures. By MRI and MRA, the anatomic information necessary for stereotactic treatment planning may be assessed with a single modality as a high-resolution digital image volume. The adequacy of MR as a modality for stereotactic guidance is demonstrated by the example of the radiosurgical treatment of cerebral angiomas. An optimized acquisition protocol, a stereotactic apparatus, a distortion correction and minimization method, and a 3D treatment planning workstation are elements of a proposed preoperative approach which in a clinical study met with good acceptance.

Algorithms↗
PubMed2006

Clinical evaluation of inhaled insulin.

Diabetes affects over 18.2 million individuals in the United States alone. Current therapy to treat type 1 diabetes relies on subcutaneous insulin administration either by injection or continuous infusion. In addition, patients with type 2 diabetes who fail lifestyle intervention and oral therapy require subcutaneous insulin. Optimal injection protocols to achieve tight metabolic control often prove burdensome to patients. Thus, development of pulmonary insulin delivery to supplement and/or replace subcutaneous insulin injections may be an effective alternative, allowing patients to achieve intensive diabetes management. This review will discuss the devices in development for the delivery of inhaled insulin. In addition, the efficacy of inhaled insulin in both type 1 and type 2 diabetic populations will be discussed. Finally, the available safety data with respect to the unique pulmonary effects of inhaled insulin will be covered.

Administration, Inhalation↗
PubMed2004

A comparison of combined arthroscopic triangular fibrocartilage complex debridement and arthroscopic wafer distal ulna resection versus arthroscopic triangular fibrocartilage complex debridement and ulnar shortening osteotomy for ulnocarpal abutment syndrome.

PURPOSE: Treatment of ulnocarpal abutment (UAS) syndrome involves decompression of the pressure and impingement, or abutment of the ulnocarpal articulation. Debridement of triangular fibrocartilage complex (TFCC) tears alone in the patient with UAS may have a failure rate of as much as 25% to 30%. Ulnar shortening osteotomy (USO) can be an effective treatment of failed TFCC debridement. Good results have been reported with combined arthroscopic TFCC debridement and mechanical arthroscopic distal ulnar resection. Similar results have been reported with both ulnar shortening osteotomy and open wafer distal ulnar resections in the UAS patient. Because all of these treatment choices appear to yield similar relief of symptoms, determination of the optimal treatment protocol remains a point of debate. The purpose of this study was to evaluate 2 different surgical treatments for UAS. TYPE OF STUDY: Retrospective review. METHODS: Eleven combined arthroscopic TFCC debridement and arthroscopic distal ulna resections (arthroscopic wafer procedures; AWP) were compared with 16 arthroscopic TFCC debridement and USOs. All patients had diagnostic wrist arthroscopy and arthroscopic TFCC debridement. All patients presented with ulnar wrist pain or neutral or positive ulnar variance, and all experienced at least 3 months of failed conservative management. RESULTS: At mean follow-up times of 21 and 15 months, respectively, 9 of 11 patients showed good to excellent results after arthroscopic TFCC debridement and AWP compared with 11 of 16 after arthroscopic TFCC debridement and USO. A statistically significant difference (P <.05) in the complication rates was identified, including secondary procedures and tendonitis. One secondary procedure and 2 cases of tendonitis were seen in the arthroscopic wafer group. CONCLUSIONS: Combined arthroscopic TFCC debridement and arthroscopic wafer procedure provides similar pain relief and restoration of function with fewer secondary procedures and tendonitis when compared with arthroscopic TFCC debridement and USO, for the treatment of UAS. LEVEL OF EVIDENCE: Level III.

Adult↗
PubMed2006

Age- and gender-related differences in ischemia/reperfusion injury and cardioprotection: effects of diazoxide.

BACKGROUND: Recent studies have demonstrated that aging is associated with reduced tolerance to ischemia and that the aged (not senescent) female heart has greater susceptibility to ischemia as compared with the aged male heart. Previously, we have shown that ischemia can be modulated with cardioplegia in the male heart; however, efficacy in the female heart was unknown. METHODS: In this study, male and female mature (15 to 20 weeks) aged (>32 months) rabbit hearts (n = 134) were subjected to Langendorff perfusion. Control hearts were perfused for 180 minutes. Global ischemia hearts received 30 minutes of equilibrium, 30 minutes of global ischemia, and 120 minutes of reperfusion. Cardioplegia +/- diazoxide was infused separately, 5 minutes before global ischemia. RESULTS: Global ischemia significantly decreased postischemic functional recovery and significantly increased infarct size in the mature and aged male and female heart (p < 0.05 versus control). The effects of global ischemia were significantly exacerbated (p < 0.05) in the aged heart as compared with the mature heart. Cardioplegia +/- diazoxide significantly increased postischemic functional recovery and significantly decreased infarct size in mature male and female hearts, but these effects were significantly decreased in the aged heart (p < 0.05) and in the aged female as compared with the aged male heart. CONCLUSIONS: Postischemic functional recovery and infarct size are affected by age but not by gender. The cardioprotection afforded by cardioplegia is affected by age and gender with a strong age-by-gender interaction for end-diastolic pressure and infarct size. Our results indicate that currently optimized cardioplegia protocols effective in the male heart are not as efficacious in the aged female heart.

Aging↗
PubMed2003

Functional in vivo gene transfer into the myofibers of adult skeletal muscle.

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.

Animals↗
PubMed2005

Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K.

Endogenous cysteine proteases were given much attention lately, as their role in a variety of pathophysiological disorders became evident. Amongst them cathepsins, which are thought to be implicated in mediation of osteoporosis, cancer progression, atherosclerosis, and many other conditions, are of considerable interest as drug targets. In the presented work, papain was chosen as a model cysteine protease and panning protocol was optimized for selection of papain-binding phage-displayed peptides from a commercially available combinatorial peptide library. Different selection strategies were applied in order to select high-affinity binders. Ultimately, five cyclic peptides (CNWAAGYNCGGGS-NH2, CWSMMGFQCGGGS-NH2, CWEWGGWHCGGSS-OH, CNWTLGGYKCGGGS-NH2 (all cyclized through formation of intramolecular disulphide bond), and GNWTLGGYKGG (cyclized head-to-tail)) were synthesized and tested for inhibitory activity towards papain and human cathepsins L, B, H, and K. The peptides possess inhibitory constants in the low micromolar to mid-nanomolar range and exhibit certain selectivity for different lysosomal cysteine proteases included in this study.

Amino Acid Sequence↗