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PubMed2004

Novel magnetic supports for small molecule affinity capture of proteins for use in proteomics.

Magnetic supports are tested for use in batch affinity capture of proteins. Two types of magnetic polymer composites were used for solid phase synthesis and for the batch affinity chromatography of folate binding protein from a protein mixture. Gly-Gly-L-Methotrexate as well as other analogs were synthesized on magnetic supports consisting of either polyoxyalkyleneamine grafted onto polystyrene beads or a copolymer of polyethylene glycol dimethylacrylamide (PEGA). Both supports incorporated within their matrix sub-micron particles of paramagnetic magnetite. The peptide-methotrexate analogs were attached to the magnetic supports via a photocleavable linker. The bound methotrexate-peptide analogs were equilibrated with a protein mixture consisting of bovine albumin, chicken albumin, folate binding protein, lysozyme, lactoferrin and lactoperoxidase precursor in phosphate buffered saline (PBS) and then after magnetically separating and washing the supports of any unbound components the bound protein was removed either through the photocleavage of the tethered methotrexate-peptide ligand or via exchange with soluble methotrexate. In all cases, the photocleavage or exchange with soluble methotrexate released folate binding protein as the major affinity captured protein. Of the two magnetic supports tested, the PEGA based support was found to be superior to the polyoxyalkyleneamine grafted polystyrene support and comparable to beaded agarose in releasing bound folate binding protein. Of the two methods for removing bound protein, photocleavage of the covalently attached ligand was found to release exclusively folate binding protein as opposed to exchange with soluble methotrexate which released residual amounts of the non-specifically bound proteins bovine and chicken albumin, in addition to folate binding protein. Thus, use of the PEGA based magnetic support in conjunction with a photocleavable linker should help facilitate the automation of multiple parallel affinity chromatography for proteomics applications.

Acrylamides↗
PubMed2005

Inflammatory C-reactive protein and cytokine levels in asymptomatic people with chronic spinal cord injury.

OBJECTIVE: To determine the relation between serologic markers of information and clinical characteristics of people with chronic spinal cord injury (SCI). DESIGN: Cross-sectional study. SETTING: Academic medical center SCI outpatient clinic. PARTICIPANTS: Convenience sample of 37 men with chronic SCI and 10 healthy control subjects. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and C-reactive protein (CRP). RESULTS: The following results achieved statistical significance at P less than .05. Asymptomatic chronic SCI patients differed from referent controls with respect to serum CRP levels but not IL-6 or TNF-alpha. In SCI patients, higher levels of CRP correlated negatively with hemoglobin and albumin levels. A longer time since injury correlated with lower TNF-alpha values, whereas higher TNF-alpha levels correlated with higher serum albumin. Pressure ulcers and indwelling urinary catheters were associated with higher mean levels of CRP but not of the cytokines TNF-alpha and IL-6. Intermittent urinary catheterization was associated with lower levels of CRP when compared with other methods of bladder management. CONCLUSIONS: Asymptomatic people with long-term SCI, especially those with indwelling urinary catheters, showed serologic evidence of a systemic inflammatory state. There was no evidence of an elevation in proinflammatory cytokines. Detection of an ongoing systemic inflammatory response in apparently healthy people with indwelling urinary catheters and small skin ulcers further supports the aggressive pursuit of catheter-free voiding options and pressure ulcer healing.

Adult↗
PubMed1976

Interrelations of matrix lipids, vesicles, and calcification.

When calcifying tissues were extracted with hot pyridine or hot benzene and then decalcified and stained with Sudan black B, the areas where mineralization was being initiated stained strongly, the rest of the calcified tissues being unstained. Histochemical methods showed that lipids were responsible for the staining. They were isolated biochemically and found to be phospholipids, very resistant to extraction before decalcification of the tissues and consisting predominantly of phosphatidyl serine and phosphatidyl inositol. It was proposed that these phospholipids were active at nucleating sites in apatite crystal formation, since it is known that phosphatidyl serine binds calcium strongly. It has been shown that phosphatidyl serine is present in matrix vesicles.

Animals↗
PubMed1997

Incentive spirometry performance. A reliable indicator of pulmonary function in the early postoperative period after lobectomy?

OBJECTIVES: The purpose of our study was to validate the incentive spirometry (IS) as a simple mean to follow pulmonary function at the bedside after lung surgery. MATERIALS AND METHODS: We studied prospectively 19 patients (16 men, 3 women; mean +/- SE age, 60 +/- 2.8 years) undergoing lobectomy for lung cancer. All the patients had an obstructive pattern with FEV1/FVC below 75%. Lung volumes, including functional residual capacity (FRC) and residual volume (RV), measured using spirometry and the helium dilution technique, and IS were measured preoperatively and postoperatively at days 1, 2, 3, and 8, and at 2 months. RESULTS: Our results showed that in the postoperative period after lung resection, IS performance was well correlated (R) during the first 8 postoperative days with vital capacity (VC) (R between 0.667 and 0.870) mainly due to the excellent correlation with the inspiratory reserve volume (IRV, R between 0.680 and 0.895) but was poorly correlated with expiratory reserve volume (R below 0.340), RV (R below 0.180), and FRC (R below 0.470). CONCLUSIONS: IS can be used as a simple mean to follow lung function, especially VC and IRV, in the postoperative period in spontaneously breathing patients. IS is noninvasive and can be performed repeatedly at the bedside in the intensive care setting.

Expiratory Reserve Volume↗
PubMed1991

[The effect of nifedipine on hemodynamics and gas exchange in dogs with experimental acute respiratory insufficiency].

PURPOSE: Evaluate the action of nifedipine, a calcium channel blocking agent, on the hemodynamics and gas exchange experimental acute respiratory failure. METHODS: Lung injury was provoked in sixteen mongrel dogs with intratracheal instillation of hydrochloric acid (HC1) (0.1N; pH = 2.0; 2.0 ml/kg body weight). As steady state was achieved after HC1 instillation (maintenance of a stable arterial PO2), saline 1 ml (six dogs) or nifedipine (ten dogs) 30 micrograms/kg for body weight were intravenously injected. The hemodynamic variables and gas exchange parameters were analyzed before HC1, after HC1 and 10 and 30 minutes after nifedipine or saline. RESULTS: The intratracheal instillation of HC1 provoked significant drop of PaO2, of systemic oxygen transport index (ITO2S), and increase of venous admixture (QVA/Q). Nifedipine provoked significant reduction of the mean systemic arterial pressure (Pas), and of the systemic (IRVS) and pulmonary vascular resistance index (IRVP), with significant increase of cardiac (IC) and systolic index (IS), with no changes ot the mean arterial pulmonary (Pap) and capillary pressures (Pcap). After nifedipine there was a significant increase of PaO2, PvO2, and ITO2S, with no significant variations of QVA/Q and alveolar arterial O2 difference (P(A-a)O2). CONCLUSION: Nifedipine promoted systemic vasodilation, and probably by increasing the venous return and/or by a reflex mechanism, the cardiac output increased, augmenting the ITO2S. The IRVP decreased in the nifedipine group, with no significant alterations of Pap and Pcap, probably consequent to the systemic vasodilation provoked by the drug. The arterial PO2 augmented in the nifedipine group, as a consequence of mixed venous PO2 increase, since no changes occurred in QVA/Q, P(A-a)O2, inspired fraction of O2 and alveolar ventilation.

Acute Disease↗
PubMed1979

The heat produced by frog muscle in a series of contractions with shortening.

1. Heat production has been measured in muscles undergoing rapid shortening 1 sec after the start of stimulation in a 2 sec tetanus. In a series of three such tetani where the periods of shortening were separated by 5 sec the shortening heats in the second and third tetani were 111 (+/- 4)% and 116 (+/- 6)% respectively of that in the first tetanus (mean +/- S.E. of mean, nine experiments). 2. When a similar series was carried out after an interval of 3 min the shortening heat in the first tetanus of the second series was 127 (+/- 3)% of that in the first tetanus of the first series (mean +/- S.E. of mean, three experiments). 3. The results are not in agreement with those of Dickinson & Woledge (1974). The discrepancy is explained by an artifact which, with certain arrangements of the apparatus, affects the measurements of shortening heat in a series of contractions. We have measured this artifact by two independent methods; the shortening heat values quoted above have been corrected accordingly. 4. The increase in the shortening heat in repeated contractions is not a specific consequence of previous shortening; an increase of the same magnitude is observed following isometric tetani. 5. During the time when the muscle is shortening, the total rate of heat production, which is initially high, falls to a steady value. This value is not significantly different in the three tetani of the series.

Animals↗
PubMed2004

Conjunctival metastasis as initial sign of disseminated cutaneous melanoma.

PURPOSE: To describe a patient with conjunctival metastasis as the initial manifestation of metastasis from a cutaneous melanoma. DESIGN: Single interventional case report. METHODS: A 48-year-old woman with a history of cutaneous axillary melanoma developed a rapidly growing conjunctival mass. Subsequent systemic evaluation disclosed asymptomatic liver metastasis. The conjunctival lesion was resected. RESULTS: Histopathologic evaluation of the conjunctival tumor disclosed an epithelioid cell melanoma located in the conjunctival stroma, without appreciable junctional activity, compatible with metastatic melanoma. CONCLUSIONS: Conjunctival metastasis from cutaneous melanoma can rarely be the initial manifestation of disseminated melanoma.

Conjunctival Neoplasms↗
PubMedDate not supplied

Evaluation of immunoassays for cannabinoids in urine.

A comparison was made of several cannabinoid urine assays. Two hundred randomly selected urine specimens were initially screened by two enzyme immunoassays (EMIT-st and EMIT-d.a.u.) and a radioimmunoassay (Abuscreen RIA). Selected specimens found positive by any of these methods were further analyzed by gas-liquid chromatography with flame ionization detection (GLC/FID), gas chromatography/mass spectrometry (GC/MS), and an experimental RIA from Research Triangle Institute (RTI RIA). The GLC/FID method gave confirmations in 69 to 92% of the samples, depending on the method used and the cut-off employed. GC/MS confirmed 98% of the EMIT and RIA positives using a low cut-off (20 ng/mL). All RIA positives at 100 ng/mL were confirmed by GC/MS. There was complete agreement between the RTI RIA and the EMIT assays, but not with the Abuscreen RIA at the 100 ng/mL cut-off. The study illustrates that care must be exercised in establishing assay cut-offs and the designation of false positive results.

Cannabinoids↗
PubMed1988

Drug testing in the military--technical and legal problems.

The Armed Forces drug-testing program has been in operation since 1971. Since that time, its goals, capabilities, and methods of operation have undergone extensive evolution and change. As these laboratories developed, they helped to establish many of the currently accepted operating standards for drug screening. For laboratories considering entry into the business of drug screening, an examination of the evolution and current operation of the military laboratories will be of benefit. It will help to define the currently accepted standards and NIDA guidelines and assist them to avoid the types of problems that were encountered by the military laboratories.

Gas Chromatography-Mass Spectrometry↗
PubMed2004

The use of specimens from various genitourinary sites in men, to detect Trichomonas vaginalis infection.

Variations in estimates of prevalence of trichomoniasis in men may reflect true differences in the burden of disease but are also affected by the performance of diagnostic methods and the type of specimen tested. In this study, men were evaluated at baseline and at follow-up, to evaluate syndromic management of urethritis and the effects of human immunodeficiency virus and trichomoniasis, in Lilongwe, Malawi. First-void urine specimens and urethral swabs were obtained at enrollment, for Trichomonas vaginalis culture; semen specimens were also obtained at follow-up. The sensitivities of testing methods using urine specimens and urethral swabs were equal; 67% of cases were identified by use of either specimen, and, in 47% of cases, both specimens tested positive. When semen specimens were included, all 3 specimens tested positive in only 19% of cases. Semen was the most sensitive single specimen, and, in 25.6% of cases, only semen specimens tested positive. Thus, prevalence of T. vaginalis infection in men is underestimated if only 1 specimen is tested.

Adolescent↗
PubMed2004

Vesicovaginal fistula after Irving sterilization: a case report.

BACKGROUND: Patients scheduled for gynecologic surgery must be informed about the development of urogenital fistulae as a possible complication. CASE: A 49-year-old woman underwent Irving sterilization with subsequent abdominal hysterectomy due to intraabdominal abscess formation. Inadvertent damage complication to the bladder during hysterectomy led to development of a vesicovaginal fistula despite intraoperative management. The fistula was repaired at once in a second session. As a sequela of bladder injury, the patient had recurrent vesical diverticula and urolithiasis with repeated removal of the diverticula and stones. A subsequent intervention performed laparoscopically led to development of a second vesicovaginal fistula. In addition, an anesthesia-induced complication resulted in cerebral hypoxia with postoperative development of Parkinson's disease. CONCLUSION: Retrospectively, there is doubt whether the proper surgical procedures were employed in this case. The fact that the patient had type 1 diabetes mellitus gives rise to the question of why no conservative method of contraception was chosen. The type of operative approach in elective surgery should always be planned on the basis of the individual patient's overall condition and in relation to the anatomic situation.

Abdominal Abscess↗
PubMed2003

Selective expression of glutathione S-transferase genes in the murine gastrointestinal tract in response to dietary organosulfur compounds.

A short-term feeding regimen was designed to analyze the effects of compounds such as diallyl disulfide (DADS), diallylthiosulfinate (allicin) from garlic and butylated hydroxyanisole (BHA) on glutathione S-transferase (GST) expression in the gastrointestinal tract and liver of male mice. After animals were force-fed these compounds, tissue GSTs were purified and individual subunits resolved by HPLC and identified on the basis of mass spectrometry (ESI MS) and immunoreactivity data. The effects of DADS and allicin on GST expression were especially prominent in stomach and small intestine, where there were major coordinate changes in GST subunit profiles. In particular, the transcripts of the mGSTM1 and mGSTM4 genes, which share large segments of common 5'-flanking sequences, and their corresponding subunits were selectively induced. Levels of alpha class subunits also increased, whereas mGSTM3 and mGSTP1 were not affected. The inducible mGSTA5 and non-responsive mGSTM3 subunits had not been identified previously. Liver and colon GSTs were also affected to a lesser extent, but this short-term feeding regimen had no effect on GST subunit patterns from other organs, including heart, brain and testis. Real-time PCR (TaqMan) methods were used for quantitative estimations of relative amounts of the mRNAs encoding the GSTs. Effects on the transcripts generally paralleled changes at the protein level, for the most part, however, the greatest relative increases were observed for those mRNAs that were expressed at low abundance constituitively. Mechanisms by which the organosulfur compounds operate to affect GST transcription could involve reversible modification of certain protein sulfhydryl groups, shifts in reduced glutathione/oxidized glutathione ratios and resultant changes in cellular redox status.

Allyl Compounds↗
PubMed1989

Fluid distribution in pork, measured by x-ray diffraction, interference microscopy and centrifugation compared to paleness measured by fiber optics.

Moderately PSE (pale, soft, exudative) and moderately DFD (dark, firm, dry) pork was examined by x-ray diffraction for interfilament separation, by differential interference contrast microscopy for interfiber area, and was centrifuged to measure water holding capacity (WHC). Internal reflectance spectra were measured by fiber optics. For PSE to DFD pork, filament separation ranged from 39 to 48 nm, interfiber area from 42 to 3%, and WHC from 49 to 64%, respectively. The correlation of reflectance with interfilament separation varied considerably with wavelength (reaching r = -.83 at 680 nm, P less than .005). The correlation of reflectance with interfiber area was more uniform across the spectrum (reaching r = .90 at 450 nm, P less than .005), as was the correlation of reflectance with WHC (reaching r = -.80 at 400 nm, P less than .005). At 24 h postmortem, fiber-optic spectrophotometry may be used as a rapid, nondestructive method to predict WHC and potential fluid losses from commercial pork with a moderate range from PSE to DFD. Interfiber area was correlated negatively with filament lattice area and WHC, but no significant correlation was found between filament lattice area and WHC. Filament separation was decreased only slightly by centrifugation. These results indicate that at 24 h postmortem the extra fluid released from PSE pork already has been lost from the myofilament lattice and is awaiting release from compartments downstream such as interfiber and interfascicular spaces.

Animals↗
PubMed2026

Phase 3 Trial of Oral Infigratinib in Children with Achondroplasia.

BACKGROUND: Achondroplasia is a genetic skeletal condition caused by FGFR3 pathogenic variants. Infigratinib, an oral FGFR1-3 tyrosine kinase inhibitor, down-regulates key pathways in the pathogenesis of achondroplasia. METHODS: In this phase 3, multicenter, double-blind, placebo-controlled trial, we randomly assigned children with achondroplasia (3 to 17 years of age) in a 2:1 ratio to receive infigratinib (at a dose of 0.25 mg per kilogram of body weight) or placebo once daily for 52 weeks. The primary end point was the change from baseline in the annualized height velocity in the infigratinib group as compared with the placebo group at week 52. Key secondary end points were the change from baseline in the height z score and in the upper-to-lower body segment ratio at week 52. The primary analysis evaluated the treatment effect at week 52 in the full analysis population, with missing data handled with a prespecified imputation approach. RESULTS: In all, 114 patients underwent randomization: 75 patients to receive infigratinib (with 1 withdrawal before treatment) and 39 patients to receive placebo. The difference between infigratinib and placebo in the least-squares mean change from baseline to week 52 was 1.74 cm per year (95% confidence interval [CI], 1.31 to 2.17; P<0.001) for the annualized height velocity, 0.32 (96% CI, 0.23 to 0.41; P<0.001) for the height z score, and -0.02 (96% CI, -0.06 to 0.01) for the upper-to-lower body segment ratio. Adverse events occurred in 71 of 74 patients (96%) in the infigratinib group and in 37 of 39 patients (95%) in the placebo group; serious adverse events occurred in 4 of 74 patients (5%) and 1 of 39 patients (3%), respectively. No serious adverse events or adverse events leading to treatment discontinuation were considered by the investigator to be related to infigratinib or placebo. CONCLUSIONS: In children with achondroplasia, treatment with once-daily oral infigratinib for 52 weeks resulted in a significantly greater increase from baseline in the annualized height velocity than placebo. (Funded by BridgeBio Pharma; PROPEL 3 ClinicalTrials.gov number, NCT06164951; EudraCT number, 2023-506130-67.).

Adolescent↗
PubMed2006

Clonal analysis of mouse development reveals a polyclonal origin for yolk sac blood islands.

Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event.

Animals↗
PubMed2005

Cellular therapy for disc degeneration.

STUDY DESIGN: Review article regarding the developing field of cellular therapies for symptomatic disc degeneration. OBJECTIVE: To review the rationale and discuss the results of cellular strategies that have been proposed or investigated for disc degeneration. SUMMARY OF BACKGROUND DATA: Disc degeneration is a substantial clinical problem. Disc degeneration begins with a loss of disc cells and alterations in the extracellular matrix of the disc. One promising approach for this problem involves the use of cells transplanted to the degenerative disc to achieve functional tissue repair. METHODS: The rationale for using cellular therapy for disc degeneration is discussed. The basic science studies involving cellular transplantation to the disc are reviewed and future directions of this line of research are discussed. RESULTS: Although substantial work remains, the future of cellular therapies for symptomatic disc degeneration appears promising. CONCLUSION: Continued research is warranted to further define the optimal cell type, scaffolds, and adjuvants that will allow successful disc repair in human patients.

Animals↗
PubMed2001

Variations of intracavitary applicator geometry during multiple HDR brachytherapy insertions in carcinoma cervix and its influence on reporting as per ICRU report 38.

PURPOSE: This paper examines the extent of variation in the applicator geometry during multiple high dose rate (HDR) intracavitary brachytherapy (ICBT) applications and its impact on reporting as per ICRU report 38. MATERIALS AND METHODS: Eighty orthogonal radiographs from 20 consecutive patients of carcinoma cervix (FIGO stages, IIA-IIIB) having four HDR ICBT applications of 6 Gy each at weekly intervals following teletherapy were evaluated. The applicator consisted of a flexible intrauterine tandem (IUT) independent of the ovoid assembly. The applicator geometry was evaluated in terms of: alpha angle, beta angle, intrauterine length (IUTL), interovoid (IOV), os to right ovoid (ORT) and os to left ovoid (OLT) distances along with vertical (VDL) and anteroposterior displacements (ADL) of the os with respect to the ovoids. The Cartesian co-ordinates (X, Y, and Z) of the IUT tip, centre of both ovoids and os were also measured. Doses to right point A (ARD), left point A (ALD), along with a reference volume of 6 Gy for ICRU height (IRH), width (IRW), thickness (IRT) and volume (IRV) were estimated for each application. RESULTS: Highly significant differences (P<0.001) between four insertions in any given patient across 20 patients for alpha angle, beta angle, IUTL, IOV, ORT, VDL, co-ordinates of the IUT, ovoids and os were observed, except for ADL (P=0.041) and OLT (P=0.247). As a consequence, variations were observed in ARD (P=0.027), ALD (P=0.017); IRH, IRW, IRT and IRV (all P<0.001). Applicator factors which influenced the various dose specification parameters were: beta angle and ORT for both ARD and ALD; UTLN, VDL and ORT for IRH; UTLN and IOV for IRW; UTLN for IRT and VDL for the 6 Gy IRV. CONCLUSIONS: A significant variation of the applicator geometry and its movement was observed in patients undergoing multiple HDR ICBT. This could have implications for reporting dose and volume specifications as required by ICRU report 38.

Brachytherapy↗
PubMed2003

An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells.

STUDY DESIGN: The goal of this study was to develop a methodology to maintain intervertebral discs in organ culture, thereby preserving tissue architecture and metabolic function in a three-dimensional environment. METHODS: Using a microdissection technique, intervertebral discs were removed from rat lumbar vertebrae. The discs were maintained in organ culture, and cell viability was evaluated histochemically and using probes that measured mitochondrial function and thiol status. The biosynthetic activity of the cells was evaluated by Western blot and RT-PCR analysis. RESULTS: The in vitro organ culture system maintained the vitality of the nucleus pulposus cells. Cells exhibited a high membrane potential for 1 week. When cells were exposed to carbonyl cyanide 4-trifluoromethoxy phenylhydrazone, a known protonophore, the fluorescence was lost, indicating that the staining was specific for viable cells. In many cells, Celltracker Green, probe for reduced thiols, colocalized with the membrane potential. Histologic studies revealed that in culture for 1 week, normal nucleus pulposus structure was maintained; after this time period, alterations were observed. We evaluated the two tissues for characteristic phenotypic markers HIF-1alpha and MMP-2. We noted that the nucleus pulposus expressed these proteins. The RT-PCR profile at 7 days indicated that the cells also expressed collagen type II, aggrecan, and decorin. DISCUSSION: Three factors contributed to success in maintaining the vitality of the nucleus pulposus in vitro. First, the cells were confined within the disc itself; second, the medium was hyperosmotic; third, the medium was supplemented with transforming growth factor-beta. The fluorescence measurement provided a rapid method for evaluation of the status of nucleus pulposus cells. Histologic analysis confirmed that the cells remained viable for at least 1 week. Viability in terms of biosynthetic activity was further confirmed using RT-PCR and Western blot analysis. We conclude that short-term intervertebral disc organ culture can be used as a suitable in vitro model to study effects of environmental factors linked to disc degeneration and/or regeneration.

Animals↗