Interferon γ is a STAT1-dependent direct inducer of BCL6 expression in imatinib-treated chronic myeloid leukemia cells.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Mapping of electrostatic potential in deep submicron CMOS devices by electron holography.

Quantitative two-dimensional maps of electrostatic potential in device structures are obtained using off-axis electron holography with a spatial resolution of 6 nm and a sensitivity of 0.17 V. Estimates of junction depth and variation in electrostatic potential obtained by electron holography, process simulation, and secondary ion mass spectroscopy show close agreement. Measurement artifacts due to sample charging and surface "dead layers" do not need to be considered provided that proper care is taken with sample preparation. The results demonstrate that electron holography could become an effective method for quantitative 2D analysis of dopant diffusion in deep-submicron devices.

Structural basis for the activity of pp60(c-src) protein tyrosine kinase inhibitors.

Conformational searches on three closely related pp60(c-src) protein tyrosine kinase inhibitors of varying potencies were performed to determine a structural basis for their activity. The first was a linear peptide (PDNEYAFFQf), the second its 10-membered cyclic analogue, and the third a cyclic analogue with a para carboxyphenylalanine in place of one the F residues. A common backbone conformation with an antiparallel beta-sheet-like geometry capped by similar beta-turns was found for all three peptides, which may be a binding conformation and gives a candidate pharmacophore for further testing. The interaction between some polar side chains and between some of the aromatic rings may be important for maintaining the correct conformation. The differences in potencies of these inhibitors may be attributed to certain thermodynamic and chemical reasons.

Unique presentation of a bronchial foreign body in an asymptomatic child.

A child who aspirates a foreign body may present in many ways. We present the case of an 8-year-old boy whose initial presentation consisted only of his preoccupation with his lost toy and his gesturing toward his oral cavity. There was no witnessed coughing or choking episode, and there were no initial physical examination findings of note, and no abnormalities on appropriate radiographic studies. The patient was transferred to our institution for further evaluation and management. Our careful examination revealed a slight wheeze at the left upper lobe. Endoscopy was immediately performed, and bronchoscopy revealed a yellow, tubular, plastic foreign body in the left main bronchus through which the patient was breathing freely. This was removed without incident by means of optical graspers. The physical characteristics of this foreign body, including its small size, relatively inert material, and large lumen, allowed this patient to present relatively asymptomatically. This case demonstrates the need for a high index of suspicion in the evaluation and management of foreign bodies in the aerodigestive tract.

The synthesis of phosphopeptides.

Phosphorylation and dephosphorylation are key events in protein expression and regulation and in signal transduction. Phosphopeptides are very useful reagents for the study of these processes, and have been used to great advantage in the study of phosphatase substrate specificity, SH2 domain ligand specificity, and protein-protein interactions. Furthermore, the advent of cell-permeable peptide carriers, such as those from the antennapedia homeodomain and the HIV TAT transcription factor, has allowed the study of intracellular events, thus underscoring the utility of these reagents. In this paper we review methods for the synthesis of phosphopeptides with the emphasis on the preparation of phosphoamino acid building blocks.

Role of laryngoscopy, dual pH probe monitoring, and laryngeal mucosal biopsy in the diagnosis of pharyngoesophageal reflux.

There is no standard for determining significant pharyngoesophageal reflux. This prospective blind comparison study compared dual pH probe studies, direct laryngoscopy, and mucosal biopsy in children without symptoms of gastroesophageal reflux who underwent airway evaluation. Significant reflux to the lower esophageal probe did not correlate with statistical significance with reflux to the upper probe. In this group of asymptomatic patients, a positive lower pH probe finding did not correlate with upper or lower esophageal mucosal inflammation. Eosinophilia in the esophageal mucosa is diagnostic of gastroesophageal reflux disease, and was seen in 5 of the laryngeal biopsies. A weak correlation was seen between positive findings at laryngoscopy and positive posterior cricoid biopsy in this group. There may be no consistent way to predict significant pharyngoesophageal reflux in asymptomatic patients. Single-probe pH testing will not predict significant pharyngoesophageal reflux with mucosal changes. Laryngoscopy and upper pH probe findings only weakly correlate with significant histologic findings. Laryngeal and posterior cricoid biopsy may be the only sensitive test for mucosal injury. Clinical trials of empiric antireflux therapy should be used to determine whether the laryngeal changes seen in these patients are reversible.

Medical and surgical treatment of pediatric dysphonia.

The production of sound for communication is the last of the prime functions of the larynx, yet it is its most salient feature. Pediatric dysphonia may be caused by a variety of general causes. These causes may be classified as infectious, anatomic, congenital, inflammatory, neoplastic, neurologic, and iatrogenic. When a child presents with hoarseness, a complete assessment is necessary. Fine points during the history and physical may help distinguish between those pathologic conditions found in these general categories.

Cyclic peptides incorporating 4-carboxyphenylalanine and phosphotyrosine are potent inhibitors of pp60(c-)(src).

The protein tyrosine kinase, pp60(c-)(src), is involved in cellular signaling and is activated during mitosis and in various tumors. We have been employing cyclic decapeptides to identify the determinants for substrate binding and phosphorylation to develop inhibitors competitive with protein substrates of Src. A structure-activity study [McMurray, J. S., Budde, R. J. A., Ke, S., Obeyesekere, O. U., Wang, W., Ramdas, L., and Lewis, C. A. (1998) Arch. Biochem. Biophys. 355, 124] revealed that, at the position 3 residues C-terminal to the phosphorylated tyrosine (Y + 3), both glutamic acid and phenylalanine gave identical K(i), K(m), and V(max) values. We hypothesized that the area of Src that binds the Y + 3 residue contains either a positively charged lysine or an arginine, capable of ionic interactions with glutamic acid or cation-pi interactions with phenylalanine. To test this hypothesis, a series of phenylalanine analogues were substituted at position 7 (the Y + 3 residue) in cyclo(Asp(1)-Asn(2)-Glu(3)-Tyr(4)-Ala(5)-Phe(6)-Phe(7)-Gln(8)-D-Phe(9 )-Pro(10)). Of these, 4-carboxyphenylalanine (4-Cpa) and phosphotyrosine resulted in high affinity peptides exhibiting K(i) values of 0.85 and 1.1 microM, respectively, 180- and 130-fold increases in potency over the parent cyclic peptide (K(i) = 150 microM). These peptides were noncompetitive with respect to ATP and competitive against the phosphate-accepting substrate, polyGlu(4)Tyr. The truncated cyclic peptide, cyclo(Phe-4-Cpa-Gln-D-Phe-Pro-Asp-Aca) (Aca = epsilon-aminocaproic acid), which did not contain tyrosine, was also a competitive inhibitor with a K(i) value of 24 microM. We conclude that these cyclic peptides bind to a positively charged area that is near the phosphate transfer region of the active site of Src but does not necessarily include the tyrosine-binding pocket. Furthermore, the 4-Cpa-containing cyclic decapeptide shows remarkable selectivity in the inhibition of Src versus the src family members Yes and Lck, as well as other protein tyrosine kinases, Ser/Thr kinases, and other ATP-utilizing enzymes.

Fragmentation and sequencing of cyclic peptides by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A series of synthetic cyclic decapeptides and other smaller cyclic peptides were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The investigated compounds were cyclized in a head-to-tail manner and contained non-proteinaceous amino acids, such as D-phenylalanine, D,L-4-carboxyphenylalanine, epsilon-aminocaproic acid, and gamma-aminobutyric acid, and were synthesized in a program to develop inhibitors of pp60(c-src) (Src), a tyrosine kinase that is involved in signal transduction and growth regulation. Post-source decay (PSD) spectra of the cyclic peptides featured abundant sequence ions. Two preferential ring opening reactions were detected resulting in linear fragment ions with an N-terminus of proline and a C-terminus of glutamic acid, respectively. MALDI-PSD spectra even permitted de novo sequencing of some cyclic peptides. Systematic studies on cyclic peptides using this method of fragmentation have not been reported to date. This work presents an easy mass spectrometric method, MALDI-PSD, for the characterization and identification of cyclic peptides.

N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor.

The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E4Y. The non-acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the peptide.

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine.

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H-Glu-Asp-Tyr-Glu-Tyr(PO3H2)-Thr-Ala-Arg-Phe-NH2, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.

Cyclic peptides as probes of the substrate binding site of the cytosolic tyrosine kinase, pp60c-src.

A series of 48 cyclic peptides based on the amino acid sequence surrounding the autophosphorylation site of pp60(c-src) was synthesized and each was tested as both a substrate and an inhibitor of this protein tyrosine kinase. Starting with cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Ala6-Arg7-Gln8-d- Phe9-Pro10) a six-amino-acid survey was performed at positions 1 through 8 to determine which positions were critical for affinity and phosphorylation and which amino acids produced the greatest activity. Our survey found that Arg7 was detrimental for binding and phosphorylation and that aromatic residues were preferred at this position. Further increases in affinity were obtained with hydrophobic residues at position 6 with the optimum for both affinity and phosphorylation being Phe. Changes on the "amino-terminal" side of Tyr4 resulted in reduced Vmax values, illustrating the requirement for acidic residues in peptidic tyrosine kinase substrates. The result of the survey was cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Phe6-Phe7-Gln8-d-Phe 9-Pro10). The change of residues 6 and 7 resulted in a 42-fold increase in affinity and no increase in Vmax. As a substrate, this peptide displayed Michaelis-Menten kinetics at saturating ATP conditions. As an inhibitor, mixed inhibition was observed. A linear version of this peptide was 13-fold less potent an inhibitor than the cyclic peptide.

Squamous cell carcinoma of the tongue associated with cinnamon gum use: a case report.

BACKGROUND: Cinnamon aldehydes found in cinnamon-flavored gums can incite mucosal alterations at points of contact with the oral mucosa. These alterations may include inflammation and epithelial proliferation, but as a rule, the changes are reversible and promptly resolve when gum-chewing activity is discontinued. METHODS: The authors report a case of a 24-year-old woman who developed a squamous cell carcinoma of the tongue following persistent and prolonged exposure to cinnamon-flavored gum. RESULTS: Several social, clinical, and histopathologic features point to the cinnamon-flavored chewing gum as a possible causal factor in the development of the patient's oral carcinoma. CONCLUSIONS: Prompt withdrawal of cinnamon products is encouraged in heavy gum chewers who develop cinnamon-related oral lesions. For those lesions which do not promptly resolve upon cinnamon withdrawal, diagnostic biopsy should be considered to exclude the possibility of a squamous cell carcinoma.

Electrocautery versus curette adenoidectomy: comparison of postoperative results.

OBJECTIVE: Identify differences in adenoidectomy performed by curettage versus electrocautery ablation. DESIGN: To receive adenoidectomy by curettage or electrocautery ablation 38 patients undergoing adenoidectomy or adenotonsillectomy were prospectively randomized. The study was completed by 24 patients. All tonsillectomy patients received electrocautery dissection of the tonsils. Preoperative and at least 1 month postoperative video-nasopharyngoscopy was performed. Video tapes were reviewed by the authors blinded to patient identity and procedure to evaluate choanal obstruction. Preoperative and postoperative lateral neck radiographs were obtained. Blood loss and postoperative complications were recorded. SETTING: A single, tertiary care pediatric facility. INTERVENTIONS: Electrocautery ablation of the adenoid was performed with suction cautery. Curettage was performed with standard adenoid curettes. MAIN OUTCOME MEASURED: A grading system for adenoid size was developed using radiographs and endoscopic parameters. The grade of preoperative and postoperative adenoid tissue was compared between the curettage and electrocautery ablation groups. Operative blood loss was compared between the groups. The postoperative course and complications were compared. RESULTS: The preoperative grade of choanal obstruction in both groups was the same. No differences could be found in the postoperative grade between the curettage and the electrocautery ablation groups. No postoperative complications were recorded in either group. The estimated blood loss in the curettage group was 54.5 ml (S.D. 50.7) while the electrocautery ablation group averaged 3.75 ml (S.D. 6.4; p = 0.0053). CONCLUSION: There are no differences in the postoperative results of adenoidectomy performed by electrocautery ablation or curettage. There are no complications recorded in either group. Estimated blood loss was lower in the electrocautery ablation group. Decreased blood loss during the procedure makes the electrocautery ablation method of adenoidectomy attractive.

The use of polyacrylamide-based peptide synthesis resins for the generation of antipeptide antibodies.

Antibodies directed against specific amino acid sequences can serve as probes for the protein molecules from which the sequence was derived, as well as affinity purification reagents. One of the major uses of synthetic peptides is for the production of such antibodies. Typically, antigens for this purpose are prepared by coupling the peptide to a macromolecular carrier such as bovine serum albumin or keyhole limpet hemocyanin. The steps for this process are peptide synthesis, peptide purification or desalting, coupling to the carrier protein, and purifying the peptide-protein conjugate. Techniques have emerged in which the amino acid sequence of interest is synthesized on a solid support using a noncleavable linkage and the resulting peptidyl-resin is injected into animals to elicit the antipeptide immune response. These procedures reduce the number of steps required to prepare the peptidyl-antigen. Water-compatible resins based on polyacrylamide have been utilized for this purpose. In this paper the composition of these supports and their use in the generation of antipeptide antibodies is reviewed.

Otolaryngic manifestations in children presenting with apparent life-threatening events.

Apparent life-threatening event (ALTE) is a term used to characterize an event of unknown cause after an infant is found limp, cyanotic, bradycardic, and/or requiring resuscitation. Like sudden infant death syndrome (SIDS), ALTE is a general term used until a precise diagnosis can be established. The relationship between ALTE and SIDS has not been clearly defined, although 7 to 15 percent of children with ALTE die of SIDS. If children with ALTE are at greater risk for SIDS, morbidity and mortality may be prevented if the underlying pathology can be identified and corrected or closely monitored. The otolaryngologist is being consulted more frequently to evaluate children who have been through an ALTE to help elucidate any underlying pathology that may have caused the near-death experience. This retrospective chart review reports the evaluation of 30 infants with ALTE requiring consultation by the Division of Pediatric Otolaryngology at the Children's Memorial Hospital in Chicago during a 3-year period. We reviewed the literature and here compare our findings with current animal models. Of the 30 children evaluated, 53% had gastroesophageal reflux, 40% had laryngeal abnormalities, 13% had tracheal abnormalities, and 10% had pharyngeal abnormalities. Thirteen percent of the children had nonotolaryngic anomalies identified during evaluation. Surgical intervention was required in 10 patients and medical treatment was used in 18. When evaluating a child with ALTE, a complete history and physical examination, evaluation for gastroesophageal reflux, assessment for upper airway obstruction by radiographs and endoscopy, and a multidisciplinary approach are recommended.

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library.

A bacteriophage peptide library containing a random 15-amino-acid insert was screened for identification of peptide sequence(s) that bind pp60(c-src). Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue. The GXXG sequence was often repeated as GXXGXXG. Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60(c-src) tyrosine kinase activity and the importance of the glycine residues within this sequence. The peptide containing glycine had a Ki of 24 microM, whereas replacing the glycines with proline increased the Ki value to 3.1 mM.

Large-pore polydimethylacrylamide resin for solid-phase peptide synthesis: applications in Fmoc chemistry.

We have synthesized a hydrophilic crosslinked aminoalkyl polydimethylacrylamide-beaded support upon which peptides have been assembled using standard Fmoc chemistry in automated batch-wise equipment. The resin was prepared by the free radical-initiated co-polymerization of N,N-dimethylacryl-amide, N,N'-bisacrylyl-1,3-diaminopropane and a functional monomer N-methacrylyl-1,3-diaminopropane hydrochlorid. After coupling of N-alpha-tert-butyloxycarbonyl-glycine (Boc-glycine), amino acid analyses gave resin loading capacities of 0.66 mmol/g. The resulting polymer was highly swollen by polar solvents including aqueous buffers and had an exclusion limit of 50 kDa for soluble proteins. This resin was found to be an excellent support for peptide synthesis using Fmoc chemistry. Typical purities of crude peptides were 80%-95%, including sequences that failed on conventional polystyrene resins.

A synthetic peptidic substrate of minimal size and semioptimal sequence for the protein tyrosine kinase pp60c-src.

We used a novel approach to determine the minimal size and semioptimal sequence of a peptide to serve as an inhibitor and/or substrate for the protein tyrosine kinase pp60c-src. The preferred amino acids surrounding tyrosine were determined by a systematic study in which we increased the length of a series of linear peptides starting from the tripeptide EYG. Using an iterative cycle, the size of the peptide was increased one residue at a time, first at the amino terminus and then at the carboxy terminus. A series of six analogs were synthesized at each position and assayed as inhibitors and substrates. The amino acids G, A, L, F, E, and K were used to semioptimize each position. The tripeptide EYG was not a substrate nor an efficient inhibitor. With increasing size of the peptide, the Ki decreased from 10.0 to 0.10 mM. The smallest peptide to serve as a substrate was a hexapeptide. The best overall peptide obtained from this method, EFEYAFF, had a Ki value of 0.13 mM with Km and Vmax values of 0.21 mM and 680 nmol/min/mg, respectively. Our best peptide was found to have higher substrate specificity than all other commerically available peptidic substrates for pp60c-src.