Identification of functional domains in murine granulocyte-macrophage colony-stimulating factor using monoclonal antibodies to synthetic peptides

Granulocyte-macrophage (GM)-CSF is an important hematopoietic cytokine that regulates proliferation and differentiation of macrophages, neutrophils, and eosinophils. In this study, we generated mAb to five synthetic peptides that correspond to regions along the murine GM-CSF molecule. The ability of anti-peptide mAb to bind to and inhibit biologic activity of murine (m) GM-CSF was determined. mAb with the highest neutralization titers were derived from mice immunized with peptide II, which correspond to amino acids 27 to 38 of mGM-CSF. Immunochemical studies showed that peptide II specifically blocked binding of anti-peptide II mAb to GM-CSF. mAb to two other peptides in the N-terminal half corresponding to residues 7 to 17 and 47 to 58, respectively, of mGM-CSF also inhibited GM-CSF-dependent proliferation and differentiation of murine bone marrow precursors for macrophages and granulocytes. Anti-peptide mAb also inhibited growth of a murine hematopoietic cell line FDCP1 and a murine T cell line HT-2, which was shown to be dependent on GM-CSF for growth in vitro. Biologic activity of both natural and recombinant mGM-CSF was neutralized by anti-peptide mAb. These findings indicate that epitopes in the N-terminal region of mGM-CSF are important for biologic activity, and the epitope defined by peptide II (residues 27 to 38) lies within a particularly important functional domain of the mGM-CSF molecule.     

Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours

We compared the screening interpretation of fluorometric analytical results for phenylketonuria (PKU) with tandem mass spectrometry (MS/MS) in filter paper blood spots collected from newborns <24 h of age. In MS/MS, both Phe and Tyr are quantified. Two hundred and eight blood spots collected from infants <24 h of age were retrieved from storage from the California newborn screening program. These samples had been categorized on the basis of fluorometric analysis as initial negative, initial positive for hyperphenylalaninemia with negative determination on recall, or initial positive for hyperphenylalaninemia and confirmed on follow up as PKU or variant hyperphenylalaninemia. The retrieved samples were analyzed in a blinded fashion using MS/MS. Correlation analysis of fluorometry vs MS/MS for Phe concentration was high, with a Pearson correlation coefficient of 0.817. When 180 micromol/L was used as the cutoff Phe concentration for MS/MS and 258 micromol/L was used as the cutoff for fluorometry, all infants with confirmed classical PKU and variant hyperphenylalaninemia were detected. MS/MS analysis reduced the number of false-positive results from 91 to 3. Simultaneous quantification of Phe and Tyr by MS/MS with the use of a cutoff Phe/Tyr molar ratio of 2.5 further reduced the number of false positives to 1. Samples from affected infants showed a discernible trend of increasing Phe concentration and Phe/Tyr molar ratio with age of collection. These results demonstrate the utility of MS/MS in the routine PKU screening of early-discharge newborns. MS/MS reduces the false-positive rate of fluorometric screening almost 100-fold because of the improved accuracy and precision of Phe measurement and simultaneous confirmation with the Phe/Tyr molar ratio. In addition to the detection of PKU, MS/MS can also detect other aminoacidopathies and disorders of fatty acid and organic acid metabolism with lower false-positive rates than other methods currently used in newborn screening programs.     

BR96 sFv-PE40, a potent single-chain immunotoxin that selectively kills carcinoma cells

We have constructed a single-chain immunotoxin composed of the carcinoma-reactive antibody BR96 and a truncated form of Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40, was expressed in Escherichia coli and localized to the inclusion bodies. We purified and identified two species of BR96 sFv-PE40, monomers and aggregates. The monomeric form was able to bind well to the BR96 antigen, a Lewisy-related antigen, while the aggregate was not. The binding affinity of the monomeric recombinant immunotoxin was 5-fold less than intact BR96 IgG, and its specificity for the BR96 antigen was confirmed by competition analysis. Monomeric BR96 sFv-PE40 was found to be extremely cytotoxic against cancer cells displaying the BR96 antigen. The cytotoxicity of the fusion protein correlates directly with antigen density on the tumor cell lines tested. The breast carcinoma cell line MCF-7, which has the highest density of BR96 antigen, was the most sensitive to BR96 sFv-PE40, with a concentration producing 50% protein synthesis inhibition of 5 pM. BR96 sFv-PE40 was found to have a t1/2 in serum of 28.5 min in athymic mice, compared to that of the chemical conjugate, chiBR96-LysPE40, which was 54 min. These data indicate that the single-chain immunotoxin BR96 sFv-PE40 is a potent inhibitor of protein synthesis in target cell lines and may be an effective agent for the treatment of cancer.     

Selective detection of carbon-13, nitrogen-15, and deuterium labeled metabolites by capillary gas chromatography-chemical reaction interface/mass spectrometry

We have applied a new chemical reaction interface/mass spectrometer technique (CRIMS) to the selective detection of 13C-, 15N-, and 2H-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The microwave-powered chemical reaction interface converts materials from their original forms into small molecules whose mass spectra serve to identify and quantify the nuclides that make up each analyte. The presence of each element is followed by monitoring the isotopic variants of CO2, NO, or H2 that are produced by the chemical reaction interface. Chromatograms showing only enriched 13C and 15N were produced by subtracting the abundance of naturally occurring isotopes from the observed M + 1 signal. A selective chromatogram of 2H (D) was obtained by measuring HD at m/z 3.0219 with a resolution of 2000. Metabolites representing less than 1.5% of the total labeled compounds could be identified in the chromatogram. Detection limits from urine of 380 pg/mL of a 15N-labeled metabolite, 7 ng/mL of a 13C-labeled metabolite, and 16 ng/mL of a deuterium labeled metabolite were determined at a signal to noise ratio of 2. Depending on the isotope examined, a linear dynamic range of 250-1000 was observed using CRIMS. To identify many of these labeled peaks (metabolites), the chromatographic analysis was repeated with the chemical reaction interface turned off and mass spectra obtained at the retention times found in the CRIMS experiment. CRIMS is a new analytical method that appears to be particularly useful for metabolism studies.     

Survival characteristics of periodontally-involved teeth: a 40-year study

Four hundred and fifty-five teeth that were judged clinically to have a questionable prognosis were observed over a 40-year span in 166 patients in a private practice. Therapy initially was debridement with oral hygiene instruction, followed by flap curettage procedures and preventive maintenance recall therapy. An assessment of compliance based on oral hygiene and recall attendance was also performed. A total of 55 (12%) teeth were lost with an average survival rate of 8.8 years prior to extraction. No teeth were lost during the first 2 years of observation. The majority of extracted teeth were maxillary second molars (38.2%), followed by maxillary first molars (25.5%), and mandibular second molars (16.4%). Tooth loss patterns appeared to be bilaterally symmetrical, with 51% of teeth lost in the right quadrants and 49% lost in the left quadrants. Indications for extraction were primarily periodontal abscesses. Teeth that were considered to have a very questionable prognosis have been retained for many years after therapy, supportive treatment, and patient compliance with recall attendance.     

Eye Movements and Phonological Parafoveal Preview: Effects of Reading Skill.

Eye movements of skilled and less skilled readers were monitored as they read sentences containing a target word. The boundary paradigm was used such that when their eyes crossed an invisible boundary location, a preview word changed to the target word. The preview could either be identical to the target word (beach as a preview for beach), a homophone of the target word (beech as a preview for beach), an orthographic control (bench as a preview for beach), or an unrelated consonant string (jfzrp as a preview for beach). Consistent with prior research, skilled readers obtained more preview benefit from the homophone preview than from the orthographic preview. The less skilled readers, however, did not show such an effect. The results indicate that less skilled readers do not use phonological codes to integrate information across eye movements. Indeed, the results also indicate that less skilled readers do not show normal preview benefit effects. (PsycINFO Database Record (c) 2010 APA, all rights reserved)