Polymerase chain reaction in polymeric microchips: DNA amplification in less than 240 seconds.

There is much interest in developing methods amenable to amplifying nucleic acids by the polymerase chain reaction (PCR) in small volumes in microfabricated devices. The use of infrared-mediated temperature control to accurately thermocycle microliter volumes in microchips fabricated from polyimide is demonstrated. Amplification of a 500-base-pair fragment of lambda-phage DNA was achieved in a 1.7-microl chamber containing a thermocouple that allowed for accurate control of temperature. While previous work showed that Taq polymerase was inactivated when in direct contact with the thermocouple, this was circumvented with the polyimide chip by the addition of polyethylene glycol as a buffer additive. This, consequently, allowed for adequate amounts of PCR product to be observed after only 15 cycles, with a total time for amplification of 240 s.

Measurement and manipulation of the partial pressure of oxygen in the rat anterior chamber.

PURPOSE: To measure the partial pressure of oxygen in the anterior chamber of the rat eye under a variety of physiological conditions. METHODS: Polarographic oxygen electrode measurements were made in methoxyflurane-anesthetized Wistar or Sprague-Dawley rats. After ketamine-xylazine or pentobarbital induction, animals were artificially ventilated with a variety of gas mixtures; gases were directed over the corneal surface during measurement of the partial pressure of oxygen in the middle of the pupil at the surface of the lens. RESULTS: The partial pressure of oxygen in the anterior chamber of the rat eye was measured as 63 +/- 9 mm Hg (mean +/- S.D. ). Breathing 100% oxygen and delivery of 100% oxygen to the cornea additively increased aqueous humor oxygen partial pressure to levels above 279 +/- 45 mm Hg with the greatest increase coming from inhaled 100% oxygen. Conversely, inhalation and subsequent transcorneal delivery of 10% oxygen reduced levels to 22 +/- 11 mm Hg. CONCLUSIONS: These results suggest that the partial pressure of oxygen in the anterior chamber is sensitive to the environment in contact with the cornea. In the rat eye, the delivery of oxygen to the anterior chamber via transcorneal diffusion may be more significant than for larger animals.

Protein and peptide separations on high surface area capillaries.

A 50 microm capillary that has been etched with ammonium hydrogen difluoride is evaluated as a separation medium for capillary electrochromatography. For a tryptic digest of transferrin, the etched capillary gave better resolution (more peaks in the overall peptide map) and longer retention than separations done under identical experimental conditions on an unetched fused-silica capillary. Resolution on the etched capillary was improved by lowering the voltage from 300 to 267 V/cm. A four-component protein sample also resulted in longer retention on an echted capillary than on an unetched fused-silica capillary that were both coated with Polybrene. After correction for differences in electroosmotic flow between the two capillaries, the calculated electrophoretic mobilities for all four proteins were lower on the etched capillary than on the unetched fused-silica capillary. The results of both the tryptic digest and protein experiments strongly indicate the presence of chromatographic effects on the etched capillary that contribute to the increased retention and improved resolution with respect to the unetched fused-silica capillary.

Application of new analytical technology to the production of a "well-characterized biological".

The role of new analytical technology in the development of the concept of a "Well Characterized Biological" is to provide suitable methodology that allows the characterization of even the most complex protein sample so that a consistent manufacturing process can be established. Glycoproteins are among the most challenging of products to characterize because of extreme sample microheterogeneity due to the carbohydrate moieties. As an example of the appropriate use of new analytical technology this review will examine the steps necessary to demonstrate that a glycoprotein is a "Well Characterized Biological". The key to characterization of complex protein samples lies in the use of appropriate combinations of the different methods that analyse the sample from substantially orthogonal and independent directions. An important advantage of capillary electrophoresis (HPCE) in this application is the complementarily of the technique with reversed phase HPLC (RPLC). Thus mixtures of variants of a polypeptide that are difficult to separate by RPLC can often be readily resolved by HPCE. Both separation techniques are well suited to the analysis of peptide maps, although RPLC is particularly powerful when used in combination with on-line electrospray mass spectrometry (ESI-MS) which allows for the effective ionization and detection of even high MW glycopeptides. In this sense the ESI-MS is an ideal detector for on-line mass detection after a RPLC separation of medium MW fragments (300 to 6000 emu) that are typically present in an enzyme digest of a protein. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) is a valuable technique for off-line characterization of CE fractions due to the high sensitivity of the method and its tolerance of samples with moderate levels of salt. The development of an effective protocol for the analysis of glycoform populations of intact glycoproteins by a combination of HPCE and off-line MALDI-TOFMS wil be demonstrated by the successful analysis of two highly heterogeneous glycoproteins, ovalbumin and Desmodus Salivary Plasminogen Activator (DSPA).

The use of multidimensional liquid-phase separations and mass spectrometry for the detailed characterization of posttranslational modifications in glycoproteins.

The goal of characterization of the proteome, while challenging in itself, is further complicated by the microheterogeneity introduced by posttranslational modifications such as glycosylation. A combination of liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) offers the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. In the current work, the combination of liquid-phase separations and mass spectrometry is demonstrated through the on-line coupling of electrospray ionization mass spectrometry (ESI-MS) and off-line coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). LC/ESI-MS yields real-time results while maintaining the separation obtained from the LC analysis. CE/MALDI TOF-MS offers high-mass detection and extremely low detection limits. The unique separation selectivity of CE relative to reversed-phase HPLC separations of the members of a glycopeptide family was used to develop an integrated multidimensional analysis achieved by the off-line coupling of LC, CE, and MALDI TOF-MS. To demonstrate the applicability of these techniques to the characterization of the heterogeneity of posttranslational modifications present in glycoproteins, we will report on the study of the glycoforms present in a N-linked site in a single-chain plasminogen activator (DSPAα1).

Management of traumatic optic neuropathy with coexistent spinal cord injury: a case report.

Traumatic optic neuropathy (TON) causes blindness of varied severity and occurs infrequently as a complication of closed head injury. A case is presented of TON that occurred in a patient who suffered complete T4 paraplegia from a motorcycle accident but in whom no severe head injury took place. In this case, high-dose intravenous methylprednisolone was begun for the spinal cord injury and repeated 24 hours later for the TON. Vision improved from near total blindness to 20/400 in the left eye (OS) and 20/130 in the right eye (OD). Two weeks later, however, the patient's vision suddenly worsened. Magnetic resonance imaging (MRI) using fat suppression confirmed a lesion along the optic nerve consistent with TON. A third course of methylprednisolone again led to improved vision. The steroids were then tapered orally over 2 weeks and the patient had no further relapses. Moderate to severely impaired vision of 20/ 400 OS and 20/130 OD continues to interfere with the patient's function and spinal cord rehabilitation program. It was concluded that a steroid taper was important in maintaining initial visual gains in this case. Awareness of TON and careful attention to the patient's clinical course can minimize deficit and maximize functional outcomes.

Analysis of recombinant DNA-derived glycoproteins via high-performance capillary electrophoresis coupled with off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

This paper describes the analysis of glycoform populations of the glycoproteins ovalbumin and Desmodus salivary plasminogen activator (DSPA alpha 1) by a combination of capillary electrophoresis (CE) and off-line matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ovalbumin has a single N-linked glycosylation site and DSPA alpha 1 has six sites for potential glycosylation, 2 N-linked and four O-linked. The conditions used for the electrophoretic separation of ovalbumin include a borate buffer system, together with a diamine additive such as 1,4-diaminobutane (DAB). An electropherogram of DSPA glycoforms could be obtained at pH 3.0 (phosphate buffer) using a bovine serum albumin (BSA) coated capillary. Fraction collection was performed by controlled application of pressure [5000 Pa (50 mbar)] for zone elution and MALDI-TOF-MS was performed on samples prepared by a 1:1 dilution with the UV absorbing matrix sinapinic acid. Both electrophoretic separations were successfully characterized by good quality mass spectra and distinct mass trends were observed for the collected fractions. It is likely that each of the collected fractions are still mixtures of glycoforms and explanation of relative mobilities or masses of different fractions is not possible at this stage. The ability to perform rapid off-line MALDI-TOF-MS of fractions from complex electropherograms will be a powerful tool to demonstrate product consistency in the manufacture of glycoprotein pharmaceuticals.

Characterization of the stable, acid-induced, molten globule-like state of staphylococcal nuclease.

Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as determined by near- and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCl results in a second transition, this one to a more structured species (the A state) with the properties of an expanded molten globule, namely substantial secondary structure, little or no tertiary structure, relatively compact size as determined by hydrodynamic radius, and the ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state at pH 2 also causes a transition leading to the A state. Fourier transform infrared analysis of the amide I band was used to compare the amount and type of secondary structure in the native and A states. A significant decrease in alpha-helix structure, with a corresponding increase in beta or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured states is proposed.

Attenuated total reflectance Fourier transform infrared analysis of an acyl-enzyme intermediate of alpha-chymotrypsin.

Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy was used to obtain signal enhancement of the spectrum of the trans-cinnamoyl-alpha-chymotrypsin acyl-enzyme intermediate. Dilute solutions (as low as 2.5 mg/ml) of enzyme or stabilized acyl-enzyme intermediate were used to form thin films on a germanium crystal surface. The secondary structure of the enzyme thin film was shown to be consistent with the native secondary structure using deconvoluted FTIR data. A novel subtraction technique was used to eliminate interfering spectra of water vapor and protein in critical regions of analysis for esters. This permitted the difference spectra of the one new ester carbonyl bond to be discerned from the 300 or so amide bonds in the protein. The results suggest that the acyl-enzyme exists in two different conformations. This study demonstrates that ir structural information of enzyme-substrate or enzyme-inhibitor complexes can be obtained with dilute protein solutions.

Characterization of protein behavior in high-performance capillary electrophoresis using a novel capillary system.

Original calculations of over a million theoretical plate efficiency for macromolecular solutes in the open tubular high-performance capillary electrophoresis experiment considered axial diffusion to be the efficiency limiting factor. In practice, interactions of biopolymers, such as proteins, with the capillary wall has had a significant impact on readily achieving high efficiencies for a wide variety of proteins. This paper reports a capillary system in which protein-surface interactions have been minimized, resulting in high efficiencies (greater than or equal to 300,000 theoretical plates). This system allows the analysis of a set of protein standards over a wide pI range at neutral pH and moderate ionic strength. The characterization of the behavior of those protein standards in this capillary system is described.

Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment.

Failure of arteriovenous communications used for chronic hemodialysis was studied during sequential 5-year periods after placement of either endogenous Brescia-Cimino (B-C) fistulas (50 patients) or polytetrafluoroethylene (PTFE, Gore-Tex) grafts (66 patients). Venous stenosis near the anastomosis was the reason for failure in 45% of PTFE grafts compared with 16% of B-C fistulas (p less than 0.001). Failure occurred, on average, 16 months after PTFE graft placement compared with 22 for B-C fistulas (p = NS). Proximal vein segments removed from five failed and two functioning PTFE graft communications were studied using light and electron microscopy and immunocytochemical techniques. All venous segments removed during surgical shunt repair exhibited a marked intimal hyperplasia. The intimal cellular component was almost exclusively smooth muscle. Accumulation of intracellular lipid droplets was not seen. Foam cells as well as extracellular lipid deposits were absent; macrophages and lymphocytes were absent from the zone of proliferation. Ultrastructural examination revealed a large proportion of extracellular matrix surrounding smooth muscle cells in the neointima. Collagen and elastin were present in the extracellular matrix, in greatest concentration deeper in the intima. Closer to the lumen, most of the extracellular volume consisted of proteoglycan. Hemosiderin was absent from the lesions as were consistent signs of luminal and intimal fibrin. Uniform intimal gradients of actin, collagen, and proteoglycan suggest that this is a steadily progressive, rather than episodic, proliferative response. These clinical and histologic observations and an analysis of hemodynamic stresses support the postulate that upstream release of platelet-derived growth factor, and possibly, shear-induced intimal injury stimulate this response. This myointimal proliferative process provides a readily accessible model of fibromuscular hyperplasia in humans; its understanding may lead to effective methods for its prevention and may provide clues to the pathogenesis of arteriosclerosis.