A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports.

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.

Multi-dimensional mapping of pyridylamine-labeled N-linked oligosaccharides by capillary electrophoresis.

A simple, sensitive and reproducible multi-dimensional capillary electrophoresis (CE) oligosaccharide mapping method is reported. The structures of 20 identified N-linked oligosaccharides have been assigned mapping positions from which co-migrating unknown oligosaccharides can be characterized. The separation protocols developed have been demonstrated to separate both charged and neutral oligosaccharides. One dimension involves electroendosmotic flow-assisted CE in a sodium acetate buffer, pH 4.0. A second dimension involves separation based on borate complexation electrophoresis in a polyethylene glycol-containing buffer. A third dimension developed specifically for neutral oligosaccharides, using a sodium phosphate buffer, pH 2.5, has been shown to resolve neutral species not able to be separated by the other two dimensions. Thus, a three-dimensional map was generated to facilitate structural characterization of these oligosaccharides.

Micellar electrokinetic chromatography of monosaccharides derivatized with 1-phenyl-3-methyl-2-pyrazolin-5-one.

Mono- and disaccharides derivatized with 1-phenyl-3-methyl-2-pyrazolin-5-one (PMP) were separated by micellar electrokinetic chromatography (MEKC), on the basis of their ability to differentially partition between an electroendosmotically driven aqueous phase and sodium dodecyl sulfate micelles. The use of a Tris phosphate buffer, pH 7.5, containing 50 mM sodium dodecyl sulfate, provided good resolution of neutral and basic monosaccharides and disaccharides. Baseline resolution was accomplished for the monosaccharides most commonly found in glycoproteins. A mass detection limit of 20 femtomoles, at a signal-to-noise ratio of three, was achieved by monitoring UV absorbance at 245 nm. The applicability of the system described to the identification and quantitation of monosaccharides obtained from carbohydrate hydrolysates from glycoproteins was investigated.

Analysis of the cocaine metabolite in the urine of patients and physicians during clinical use.

Cocaine is considered a superb anesthetic for many otolaryngologic procedures and has been shown to be positive in the urine of patients for up to 72 hours after surgery with a standard radioimmunoassay test. The standard cutoff for drug screening of benzoylecgonine, the main urine metabolite of cocaine, has been 300 ng/ml. However, the new threshold value in many laboratories is now 150 ng/ml. In review of the literature, no study has been performed that quantitates the actual level of the urine cocaine metabolite after a routine otolaryngologic procedure in both physicians and their patients with the gold standard for urine testing, gas chromatography. This study documents the quantitative level of the urine cocaine metabolite in patients and reveals that there are metabolite levels present in physicians during a single exposure, although they are below the current cutoff level that will be picked up on current screening assays. Evidence has also been presented demonstrating a cumulative effect on the benzoylecgonine levels in physicians who clinically use cocaine anesthesia more frequently; these levels can be above the cutoff level on current screening assays.

Hydrophilic-interaction chromatography of complex carbohydrates.

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

KTP laser excision of a soft palate squamous cell carcinoma: a case report.

Squamous cell carcinoma of the soft palate is an infrequently seen tumor of the oral cavity. The use of the KTP laser in the treatment of a 52-year-old man with a T2N0M0 soft palate carcinoma is reported. The natural history and treatment of this entity is reviewed. The application of tumor thickness as related to therapy of the neck and prognosis is commented on. Use of the KTP laser for resection of oral cavity lesions is felt to be a technological improvement beneficial to both patients and practitioners.

Structural determination of the essential serine and glycosylation sites of carboxypeptidase P.

Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Ki's of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.

A general approach for characterizing glycosylation sites of glycoproteins.

Recent advances in glycobiology have greatly stimulated carbohydrate research; however, improving techniques for identification and isolation of specific glycosylation sites in protein structure analysis remains a challenge. We report here a practical approach utilizing a membrane staining technique on Problott, a PVDF-type membrane, to screen glycoproteins and glycopeptides derived from enzymatic digests of glycoproteins. To improve the detection sensitivity, an amplified staining technique using biotinylated lectins, avidin, and biotinylated peroxidase was employed. In addition, we describe a micro-batch affinity binding procedure to isolate glycopeptides from complex glycoprotein enzymatic digests. These protocols allow us to start with a subnanomole quantity of glycoprotein and locate the glycosylation sites; isolate glycopeptides in a homogeneous form; and perform amino acid composition, amino acid sequence, and mass analyses on the isolated glycopeptides. The characterization of glycosylation site of a model glycoprotein, carboxypeptidase P, of which the structure is still largely unknown, has been investigated.

Dematiaceous fungal sinusitis.

Six cases of dematiaceous fungal sinusitis are reported, together with a review of 33 other cases collated from a review of the English literature. The sinusitis was more often unilateral vs. bilateral. A characteristic serpiginous hyperdense intrasinus opacification, as well as sinus expansion with bone erosion, was often seen on CT scan. MRI scan showed lucent sinus cavities on T1 and T2 weighting. A comparison of surgical treatment vs. surgery with systemic antifungal therapy revealed decreased recurrence and complication statistics in the combined therapy group. There was a trend toward increased recurrence/persistence and complications associated with invasive histologic findings and only surgical treatment, but not statistically significant. At the present time, we recommend comprehensive surgical treatment followed by systemic antifungal therapy, though clinical judgment and individualization should occur. Future studies are planned to further define the disease entity and its therapy.

Composite resection reconstruction: split-thickness skin graft--a preferred option.

In dealing with reconstruction of the oral cavity postcomposite resection, many options are available. Maximization of function with minimization of complications, physiologic sequelae, and cost must be considered. Fifty consecutive patients who underwent composite resections and were reconstructed by split-thickness skin grafts were analyzed. Factors examined included: number of blood units transfused, disease status vs. stage, length of hospital stay, complications, use of prosthetic devices for aiding in swallowing and speech production, and patient diet at discharge. This evaluation and literature review revealed that the amount of tissue resection was considered to be the most significant functional determinant, followed by maintenance of residual tissue mobility. The use of a split-thickness skin graft was believed to give excellent results for the previously mentioned parameters and is our preferred method for reconstruction of composite resection defects that do not require tissue bulk as in anterior mandible defects, anticipated mandible reconstruction, total or near-total glossectomy, or very massive defects.

Pulmonary lymphangitic carcinomatosis from hypopharyngeal adenosquamous carcinoma.

Although pulmonary lymphangitic carcinomatosis (PLC) is not uncommon for breast, bronchial, and stomach cancers, it is rarely associated with head and neck malignancy. A case of PLC is reported with the primary lesion being an adenosquamous carcinoma of the hypopharynx. A literature review is discussed, highlighting the varied radiographic picture of PLC, the possible diagnostic modalities, the proposed pathogenesis, and the treatment options. The prognosis even if antemortum diagnosis and treatment are given remains fatal, although with temporary improved quality of life and lengthened survival. There is hope that with greater awareness of the disease process an increased incidence of diagnosis may allow progress to be made in therapeutic interventions.

Interaction between L-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production.

A mixture of threonine dehydrogenase and aminoacetone synthetase will catalyze the conversion of L-threonine to glycine. The overall reaction likely involves the conversion of L-threonine, NAD+, and CoA to glycine, NADH, and acetyl-CoA. Physical separation of L-threonine dehydrogenase from aminoacetone synthetase results in the formation of aminoacetone and CO2 from their substrates. A physical interaction between threonine dehydrogenase and aminoacetone synthetase has been demonstrated by gel permeation chromatography and fluorescence polarization. Polarization of fluorescence measurements of threonine dehydrogenase and aminoacetone synthetase labeled with fluorescein isothiocyanate indicated the formation of a soluble active complex, with an apparent dissociation constant (Kd) of 5-10 nM and an apparent stoichiometry of 2 aminoacetone synthetase dimers/1 threonine dehydrogenase tetramer. Chemical experiments have identified aminoacetone as the enzymatic product of L-threonine dehydrogenase acting on L-threonine. These experiments involved trapping pyrrole derivatives, [3H]NaBH4 reduction, and coupling with plasma amine oxidase. Kinetic experiments also showed NADH, CO2, and aminoacetone to inhibit threonine dehydrogenase in a manner consistent with an ordered Bi-Ter kinetic mechanism. NAD+ is the lead substrate followed by threonine, and the products are released in the order: CO2, aminoacetone, and NADH.

Squamous cell carcinoma with positive margins. Surgery and postoperative irradiation.

The charts of patients treated surgically for squamous cell carcinoma of the head and neck were reviewed retrospectively to correlate the pathologic report of the adequacy of margins with subsequent treatment and eventual outcome. Three hundred forty-nine patients were studied. Thirty-one patients (8.8%) had positive margins. Positive margins were most commonly encountered in patients with tonsillar and hypopharyngeal lesions. Twenty-nine (94%) of the patients had stage III or IV disease. Two patients (6.4%) remain free of disease 36 months or longer following surgery. Radiation therapy was administered postoperatively to 25 patients. Sixty percent of these patients failed to achieve local-regional control, and 84% are dead of disease. When free margins of resection cannot be obtained due to anatomic limitation, postoperative radiation therapy has been unsatisfactory in our hands. Alternative treatment, such as radiation implants or chemotherapy, would appear to offer the only hope of improving the chances for long-term survival.

Mechanism-based fragmentation of coenzyme A transferase. Comparison of alpha 2-macroglobulin and coenzyme A transferase thiol ester reactions.

The plasma proteins, alpha 2-macroglobulin and complement components 3 and 4, contain an internal thiol ester involving a glutamyl and cysteinyl residue. The thiol ester is susceptible to cyclization at greater than 37 degrees C and forms an unstable 5-oxyproline intermediate. The latter can be hydrolyzed to produce two peptide fragments. We propose that enzymes having activated glutamyl residues as part of their catalytic mechanisms may undergo an analogous cyclization and peptidyl cleavage. As a model, we have investigated pig heart succinyl-CoA:3-keto acid transferase. When the CoA-enzyme thiolester intermediate is heated at pH 7.4 and 70 degrees C for 1 h, approximately 60% of the Mr = 60,000 subunits are cleaved to give Mr = 40,000 and 20,000 fragments. We have shown that formation of the enzyme thiolester is an obligate precursor for the protein fragmentation. However, the reaction was incomplete with a maximum of approximately 65% cleavage at times greater than 60 min. These results suggest that there is a competing, deactivation reaction; namely, the thiol ester and oxyproline intermediates are hydrolyzed to regenerate the active site glutamic acid. Although the maximum rate of cleavage is at 70 degrees C, approximately 15% autolysis also occurs at 37 degrees C. The Mr = 40,000 fragment had the same amino terminal sequence as the Mr = 60,000 subunit, (Trp-Lys-Phe-Tyr-Thr-Asp-Ala-Val-Glu-Ala-). No amino terminal could be detected for the Mr = 20,000 fragment, even after digesting the fragment with pyroglutaminase. Peptide maps of the fragments and the uncleaved subunit indicate that the fragments are generated in parallel. The size of the fragments puts the active site about two-thirds of the way from the amino terminal of the protein.

Decarboxylation of glycine by serine hydroxymethyltransferase in the presence of lipoic acid.

Serine hydroxymethyltransferase and the glycine cleavage system are both present in liver mitochondria and both bind glycine to form a pyridoxal 5'-phosphate carbanionic quinoid species. Lipoic acid has been shown to have the ability to intercept the carbanionic intermediate formed from the binary complex of serine hydroxymethyltransferase and glycine and form an intermediate adduct which is ultimately processed to yield CO2 and a methylamine adduct. Kinetic studies have shown that the lipoic acid-dependent decarboxylation of glycine catalyzed by serine hydroxymethyltransferase proceeds through a sequential mechanism. This lipoic acid-dependent decarboxylation catalyzed by serine hydroxymethyltransferase is similar to the initial reaction of the glycine cleavage system and to the lipoic acid-dependent decarboxylation of glycine by the P-protein alone suggesting that both enzymes could serve in lieu of each other.