Michael G. Harrington

Methods for increasing the resolution of two-dimensional protein electrophoresis

A two-dimensional gel elctrophoresis protocol has been developed which provides for a 1.5-to 3-fold increase in the resolution of proteins compared to other frequently used methods. The major variations from previous protocols include increased pore size in the isoelectric focusing gels; cholamidopropyldimethylhydroxypropanesulfonate, a zwitterionic detergent, replaces most of the Nonidet P-40, a nonionic detergent, in the isoelectric focusing gels; no equilibration step is employed between the first and second dimensional separation. The use of a stacking gel in the second dimension has been eliminated; a more efficient and evenly distributed cooling system has been designed for the molecular mass separation, allowing faster migration with higher current. Finally, the crosslinker diacrylylpiperazine is employed which improves protein separation and detection with ammoniacal silver staining. Silver-stained two-dimensional gel electrophoretograms of human plasma and hamster brain tissues and autoradiographs of rat liver cells are compared to the results obtained from previous methods.

The 14-3-3 Brain Protein in Cerebrospinal Fluid as a Marker for Transmissible Spongiform Encephalopathies

BACKGROUND There is no practical and reliable premortem test for Creutzfeldt-Jakob disease and the related transmissible spongiform encephalopathies. Two proteins, designated 130 and 131, which have been detected in low concentrations in cerebrospinal fluid from patients with Creutzfeldt-Jakob disease, appear to be sensitive and specific markers for the disease. Attempts to identify these proteins, however, have been unsuccessful. We hypothesized that they may be present in the normal brain. METHODS We detected proteins 130 and 131 in normal human brain, partially sequenced their amino acids, and found that they matched the brain protein known as 14-3-3. We then developed a simple, rapid immunoassay for this protein and tested it in cerebrospinal fluid samples from 71 humans and 30 animals with spongiform encephalopathies and in control samples from 186 humans and 94 animals. RESULTS The immunoassay detected the 14-3-3 protein in cerebrospinal fluid from 68 of the 71 patients with Creutzfeldt-Jakob disease (96 percent, 95 percent confidence interval, 92 to 99 percent). Among 94 patients with other dementias, the specificity was 96 percent. If one excludes the three patients with dementia who had strokes within one month before testing, the specificity was 99 percent. The test was positive in 12 of 24 patients with viral encephalitis. In animals the sensitivity of the assay was 87 percent and the specificity was 99 percent. CONCLUSION In patients with dementia, a positive immunoassay for the 14-3-3 brain protein in cerebrospinal fluid strongly supports a diagnosis of Creutzfeldt-Jakob disease. This finding, however, does not support the use of the test in patients without clinically evident dementia.

Abnormal Proteins in the Cerebrospinal Fluid of Patients with Creutzfeldt–Jakob Disease

We studied more than 300 cerebrospinal fluid proteins from 21 patients with Creutzfeldt-Jakob disease. We also examined cerebrospinal fluid from 100 normal controls and more than 400 patients with various neurologic disorders other than Creutzfeldt-Jakob disease. Four abnormal proteins that were identified in the patients with Creutzfeldt-Jakob disease were absent in the normal persons. Two of these proteins (Mr [relative molecular mass], 40,000; pl [isoelectric point], 5.7 and Mr 40,000; pl 5.9) were also present in some patients with multiple sclerosis, herpes simplex encephalitis, schizophrenia, Parkinsons disease, or Guillain-Barré or Behçets syndrome. Two proteins (Mr 26,000; pl 5.2 and Mr 29,000; pl 5.1) were present in all patients with Creutzfeldt-Jakob disease and in 5 of 10 patients with herpes simplex encephalitis, but in none of the other control groups. A subsequent blinded study of these cerebrospinal fluid proteins from patients with Creutzfeldt-Jakob disease, Alzheimers disease, Huntingtons disease, multi-infarct dementia, parkinsonism dementia of Guam, or the specific dementia of the acquired immunodeficiency syndrome resulted in the ability to distinguish all cases of Creutzfeldt-Jakob disease from the other types of dementia. Although the identity and origin of the abnormal spinal fluid proteins are not yet known, these preliminary results suggest that their presence may help in the diagnosis of Creutzfeldt-Jakob disease.

The search for mitochondrial inheritance of human diseases

Abstract Several diseases with non-mendelian maternal inheritance patterns are described, some of which appear to be associated with abnormal mitochondrial structure or function. Although no human disease has yet been proven to result from the inheritance of an abnormal mitochondrial genome, such defects may be implicated in maternally inherited diseases. New techniques in molecular genetics, including DNA sequencing, endonuclease restriction analysis and analysis of mitochondrial gene products, should clarify the molecular basis of these maternally inherited inborn error of metabolism.

Metabolic Engineering of N-Linked Glycoform Synthesis Systems in Chinese Hamster Ovary (CHO) Cells

Successful metabolic engineering of glycoform distribution to obtain a preferred glycoprotein product can be aided by a systems approach which considers the multiple input (enzyme and cosubstrate levels and localization-multiple output (glycoform distributions on multiple glycoproteins) nature of Oligosaccharide biosynthesis. Elements of such a systems approach now under development in our laboratories include a new mathematical framework for computer simulation of N-linked glycosylation which can be used to suggest effective glycosylation engineering strategies. Also, two-dimensional protein electrophoresis protocols for resolution of hundreds of Chinese hamster ovary (CHO) cell glycoproteins have been developed and applied to study global changes in Oligosaccharide modification in wild-type CHO and in an β-N-acetyl glucosaminyl transferase III (GnTIII)-expressing mutant CHO cell cultures. The action of GnTIII creates a bisected Oligosaccharide and reduces the in vitro activity of at least five other enzymes involved in the same glycosylation pathway, giving this enzyme an important role in controlling the biosynthesis of complex and hybrid Oligosaccharides. These studies show that many CHO glycoproteins are modified by the action of GnTIII.. Levels of several glycosyltransferases must be genetically modified in order to achieve certain glycoform distributions, such as altered patterns of Oligosaccharide branching. As an initial step towards cell Unes providing these capabilities, we have cloned GnTIII into a s-interferon (IFN-s) overproducing CHO cell line. Four individual clones produce a novel glycoform of recombinant human IFN-s containing Oligosaccharides which bind the lectin E-PHA and thus likely contain bisected structures.

Simultaneous analysis of phosphoproteins and total cellular proteins from PC12 cells

We describe a method for identifying phosphoproteins among total cellular proteins using two-dimensional electrophoresis (2DE). The method involves the simultaneous double-labeling of cells with [ 35 S]methionine and [ 32 P]orthophosphate. Proteins that incorporate one or both isotopes are then identified within the same gel on the basis of the different energies of the respective isotope emissions. Detection is achieved using a storage phosphor screen that is exposed to the dried 2DE gel with or without (+/−) the insertion of a copper filter between the gel and the storage phosphor screen. Each of these two accumulated potential images is digitized on a Phosphorimager. Because both +/− copper-filtered images are derived from the same gel, it is possible to precisely overlay the two images. Measuring the efficiency of transfer of radiation across the copper for each spot allows accurate determination of whether there is a double or single label in each protein. The dynamic range of the method allows quantitation over five orders of magnitude for a single isotope and over four orders of magnitude for 32 P and 35 S isotope mixtures in the 2DE gel. The sensitivity is more than 10-fold greater than that of autoradiography and requires exposures of 2–48 h instead of several weeks. We describe the application of this method to the identification of protein phosphorylation events in a rodent cell line (PC12 cells) after exposure to nerve growth factor. This method should have wide applicability in identifying post-translational modifications of proteins involved in many biological processes.

Cross-linked poly(N-acetylethylenimine) as an isoelectric focusing matrix

Agarose and polyacrylamide are the gels used for most analytical and micropreparative electrophoresis of biopolymers. In an alternative approach that offers different physico‐chemical properties from these standard gels, nonionic hydrogels and amphigels composed of poly(N‐acetylethylenimine) (PAEI) and a variety of cross‐linkers were prepared and used as anticonvective matrices for isoelectric focusing. PAEI was prepared from the ring opening, ionic polymerization of 2‐methyl‐2‐oxazoline. The N‐acetyl side chains were hydrolyzed with aqueous sodium hydroxide to produce secondary amine sites which were used for the attachment of cross‐linkers. Several cross‐linkers were tested for their suitability for electrophoresis, and the cross‐linker system based on the Diels‐Alder reaction between a furan and maleimide tethered to PAEI gave a moldable gel that can be reversibly converted to a sol at 80oC. This gel was used for isoelectric focusing under both denaturing and nondenaturing conditions. Several protein standards were resolved as well as was achieved with polyacrylamide.

Naturally Occurring Scrapie in Southdown Sheep

The purpose of this study was to characterize naturally occurring scrapie in the Southdown breed of sheep. Experimental subjects included 4 Southdown ewes admitted to the University of Missouri, College of Veterinary Medicine Large Animal Clinic. All 4 sheep had signs compatible with clinical scrapie. Cerebrospinal fluid (CSF) cell counts ranged from a low of 1 nucleated cell/microL to high of 4 cells/microL with a median of 3 cells/microL. Cerebrospinal protein concentrations ranged from 26 to 78 mg/dL with a median of 53 mg/dL. Immunoassay of the CSF for the 14-3-3 protein yielded positive results in 3 of the 4 sheep. Sequencing of the prion protein (PrP) gene revealed that all 4 sheep were homozygous for glutamine at codon 171 and, hence, were of the QQ genotype. Histopathologic examination of brain stem tissue sections revealed intracytoplasmic neuronal vacuolation and mild spongiform changes in the gray matter neuropil in all 4 ewes. The diagnosis of scrapie was confirmed by immunohistochemical staining for the abnormal PrP Our results suggest that the genetics of scrapie susceptibility are probably similar in Suffolk and Southdown sheep. Positive immunoassay results for the 14-3-3 protein were observed in 3 of the 4 sheep.