Separation of the two most closely related isoenzymes of alkaline phosphatase by two-dimensional electrophoresis.

Seminoma is the most frequent testicular germ cell tumor. While effective curative treatment of the disease is available today, there is to date no tumor marker suited for the diagnosis and follow-up. Several authors have suggested that the germ-cell-specific isoenzyme of alkaline phosphatase (GCAP) might be valuable for this purpose. GCAP shows 98% sequence homology with the placental isoenzyme of alkaline phosphatase (PLAP). Both display a high degree of phenotypic heterogeneity. Until now all attempts to raise an antibody reacting specifically with GCAP have failed. Consequently there is no immunological assay that allows the measurement of GCAP in the presence of PLAP. Two-dimensional electrophoresis with a sigmoid immobilized pH-gradient of 3-10 for the first dimension makes it possible to differentiate clearly between these two closely related isoenzymes. Additionally, it resolves their many phenotypic variants. This is of special interest, since malignant transformation affects the glycosylation patterns of many glycoproteins. For the detection of GCAP and PLAP in two-dimensional electrophoresis it is essential to purify the raw tissue extracts thoroughly. A chromatographic method suited for this purpose is presented.

Electrophoretic, chromatographic and immunological studies of human urinary proteins.

Urinary proteins from both sexes were analyzed by high resolution two-dimensional gel electrophoresis (2-DE). For well reproducible 2-DE patterns, the samples were concentrated and desalted in one step by vacuum dialysis. A reference map for urine proteins was established by the analysis of urine from 10 healthy persons. Proteins in urine that share immunogenicity with serum proteins were identified by use of antibody to whole human-serum protein in an affinity-column fractionation of urine and differential analysis of the adsorbed (serum component) and unadsorbed (non-serum component) fractions. For identification of individual proteins, coelectrophoresis, immunoblotting and affinity chromatography with corresponding antibodies were used. Proteins identified in the map, besides known serum proteins, included: the subunit of Tamm-Horsefall protein, the secretory component of IgA, constant breakdown products of alpha 1-antitrypsin and retinol-binding protein, the five isoforms of the beta chain of human chorionic gonadotropin and the subunit of prostatic acid phosphatase. In addition, we could demonstrate three proteins which are markedly pronounced in female urine, especially pregnant women. To get more information about the native properties of various urinary proteins, they were separated into four main peaks according to their sizes using fast protein liquid chromatography equipment. Possible interpolypeptide disulfide bonds were studied using a nonreducing 2-DE system. 2-DE in combination with other methods seems to be a valuable tool for the characterization of urinary proteins in defined renal or extra-renal diseases. An example is given by analyzing the immune complexes from seven patients with a urinary tract infection.

Immune complexes in blood: a new strategy for the analysis of their antigen portion.

A method is described for the characterization of protein antigens from circulating immune complexes from plasma. Free immunoglobulins G were separated from larger immune complexes by gel filtration with a fast protein liquid chromatographic system. The collected immune complexes were dissociated with 4M urea into antigens and antibodies. With a second column run with 4M urea, antigens smaller than 120 kDa were separated from unloaded antibody fractions. After concentration, they were analyzed by two-dimensional gel electrophoresis.

Analysis of cerebrospinal fluid proteins by electrophoresis.

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt-Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.

Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine.

The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein-A and protein-G affinity chromatography and analyzed by two-dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids.

Chromatographic and electrophoretic studies of circulating immune complexes in plasma.

The protein nature of soluble immune complexes from fresh plasma was studied by combining several analytical biochemical techniques. Free immunoglobulins (Ig) G were separated from larger immune complexes by gel permeation chromatography. In a second step, immune complexes, free IgA and IgM were isolated by protein-A and protein-G affinity chromatography and analysed by two-dimensional gel electrophoresis. Sixteen plasma samples from healthy donors were analysed and evaluated visually. Their protein profiles on the gels turned out to be similar, showing only slight quantitative differences. In one case, additional proteins were detected. To prove the ability of the method, immune complexes were analysed from four plasma samples that showed macro creatine kinase type 1, a complex formation between creatine kinase BB and IgG. This methodology can be used for the examination of immune complexes of unknown protein composition in serum or plasma.

Mechanism of imipenem resistance acquired by three Pseudomonas aeruginosa strains during imipenem therapy.

Imipenem sensitive pretherapy isolates (MICs 1-2 mg/l) and the corresponding resistant posttherapy isolates (MICs 16 mg/l) of Pseudomonas aeruginosa from three patients undergoing imipenem treatment were analyzed to establish the resistance mechanism. The identity of pyocin types, serotypes, DNA restriction endonuclease profiles and plasmid profiles strongly suggested isogenicity of pre- and posttherapy isolates. The imipenem resistant posttherapy isolates showed cross-resistance only to another carbapenem, meropenem. There were neither qualitative nor quantitative differences between pre- and posttherapy isolates in beta-lactamase production. Affinity of the penicillin-binding proteins 1A, 1B, 2, 3, 4,4' and 5 for [14C]imipenem was the same in pre- and posttherapy isolates. One-dimensional and two-dimensional gel electrophoresis of outer membrane protein preparations showed diminished expression of an outer membrane protein of about 46.5 and 47.5 kilodaltons, respectively, in the posttherapy isolates. This protein had an apparent isoelectric point of about pH 5.2 in two-dimensional gel electrophoresis. Growth in proteose peptone no. 2 broth did not reduce expression of this outer membrane protein, which spoke against its identity with the outer membrane protein D1. The permeability of the outer membrane for imipenem was reduced in the posttherapy isolates, since addition of 0.5 or 0.25 of the MIC of the permeabilizing agent ethylene-diaminetetraacetate reduced the MICs of imipenem for all isolates from each patient to the same (susceptible) level. The diminished expression of one of the outer membrane proteins might be the reason for this reduced permeability.

Analysis of immune complexes of cerebrospinal fluid by two-dimensional gel electrophoresis.

From the cerebrospinal fluid of 32 patients with different neurological diseases immune complexes were isolated using protein A-Sepharose. The isolated heavy and light chains and their constituents were analyzed by two-dimensional gel electrophoresis. In addition to immunoglobulins, some proteins such as albumin, apolipoprotein A-I and a number of unknown proteins were detected in all preparations. A complex consisting of three proteins with molecular masses between 52-55 kDa reacted slightly with polyclonal antibodies to glial fibrillary acidic protein. Whether the linkage between these antigens and the Ig is due to the Fab region or the Fc region remains unknown in our study. In some immune complexes of neurological diseases such as amyotrophic lateral sclerosis, astrocytoma and multiple sclerosis, differences are easily recognizable in the gel pattern.

Study of human cerebrospinal fluid proteins by size exclusion-high performance liquid chromatography and two-dimensional gel electrophoresis.

Cerebrospinal fluid (CSF) proteins were separated into three main fractions by size exclusion-high performance liquid chromatography (SE-HPLC). Subsequent analysis of each fraction by two-dimensional gel electrophoresis (2-DE) facilitated the detection of trace components in CSF and additionally provided more information about the native properties of various proteins. Certain proteins are present in a polymeric form and appear in the high molecular weight SE-HPLC fraction. In the middle molecular weight SE-HPLC fraction we found a CSF-specific transthyretin-related protein by immunoblotting with polyclonal antibodies to transthyretin. Possible interpolypeptide disulfide bonds of such polymeric proteins were studied using a nonreducing 2-DE system. This procedure revealed that all apolipoprotein E monomers in CSF, which are synthesized in astrocytes, are linked by disulfide bonds. In the CSF from a patient with clinically definite multiple sclerosis (MS), novel proteins appeared in the high molecular weight SE-HPLC fraction, which are obscured by other proteins if total CSF is analyzed.

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, V. The complete amino-acid sequence of the mesophilic L-lactate dehydrogenase from Bacillus megaterium.

The complete amino-acid sequence of L-lactate-dehydrogenase from the mesophilic Bacillus megaterium was determined. 92% of the 318 amino acids were established by sequence analysis of the N-terminus, of four CNBr fragments and of one fragment obtained by cleavage with BNPS-skatole. The primary structure was completed by sequencing overlapping fragments obtained by further cleavage of suitable CNBr fragments and BNPS fragments with either trypsin, endoproteinase Lys-C, o-iodosobenzoic acid or hydroxylamine. The C-terminal amino acids were determined by degradation with carboxypeptidase A. The sequence homology between lactate dehydrogenases from B. megaterium and those from other Bacilli is 59-61% and 35-37% to those from higher organisms. The high sequence homology among lactate dehydrogenases from Bacilli, adapted to different temperatures, allows comparative studies of the structural basis of protein thermostability.

Cerebrospinal fluid proteins studied by two-dimensional gel electrophoresis and immunoblotting technique.

The proteins of lumbar CSF have been investigated by two-dimensional gel electrophoresis, and their patterns have been compared with the corresponding serum protein patterns. Serum proteins in CSF have been identified by electroblotting and immunoreaction with antiserum against total human serum proteins. Proteins derived from brain have been identified with antiserum against human brain proteins. The most prominent CSF protein group has been identified as a multiple form of apolipoprotein E. The correct position of the glial fibrillary acidic protein has also been determined. The prefractionation of CSF proteins by size exclusion chromatography or by affinity chromatography followed by two-dimensional electrophoresis has facilitated the detection of trace components in CSF and the corresponding serum.

Two-dimensional gel electrophoresis, isoelectric focusing and agarose gel electrophoresis in the diagnosis of multiple sclerosis.

Two dimensional gel electrophoresis (2-DE), isoelectric focusing (IEF) and agarose gel electrophoresis (AGE) were used to examine cerebrospinal fluid (CSF) and sera from 22 patients with confirmed multiple sclerosis, 11 patients with probable multiple sclerosis and 20 control patients with non-inflammatory neurological diseases of the central nervous system (CNS). All of the 22 patients with confirmed multiple sclerosis showed abnormal patterns of oligoclonal IgG in all three methods. In the CSF from patients with probable multiple sclerosis, oligoclonal IgG was detectable in 18 percent with AGE, in 72 percent with IEF and 90 percent with 2-DE. No oligoclonal IgG was observed in subjects with non-inflammatory neurological diseases. Many artefacts in IEF, which lead to misinterpretation, are eliminated in the 2-DE system. Based on our observations and this study in particular, it is evident that some patients have IgG changes which can be detected only by 2-DE. The application of research-oriented 2-DE for routine clinical purposes is still limited by its cost and technical complexity.

Two-dimensional gel electrophoresis of cerebrospinal fluid proteins from patients with various neurological diseases.

We subjected cerebrospinal fluid (CSF) samples from over 200 patients with various neurological diseases to two-dimensional electrophoresis. The series included non-inflammatory diseases such as epilepsy, amyotrophic lateral sclerosis, and polyneuropathy; and inflammatory diseases such as multiple sclerosis and neurolues. In the resulting electrophoretograms we considered mainly the region of CSF-specific proteins and the area corresponding to the immunoglobulin light chains. The former, at Mr 35 000-38 000, shows some interesting variations from one CSF to another. Samples from patients with various brain tumors show a specific change. A zone of oligoclonal immunoglobulin light chains appeared in all CSF samples with above-normal gamma-globulin content. These oligoclonal patterns remained constant and characteristic during the course of different diseases for several patients so examined. As expected, differences appeared in the patterns of immunoglobulin light chains from one individual to another, even among a group of patients with the same disease. The extent of the correlation of certain basic patterns with certain diseases cannot yet be determined.

[2-dimensional electrophoresis of cerebrospinal fluid in various neurological patients]. / Zweidimensionale Elektrophorese des Liquor cerebrospinalis verschiedener neurologischer Patienten.

At present two-dimensional (2D) electrophoresis is the best method of resolution for separating complex protein mixtures, and this method has been successfully adapted for resolving CSF proteins. CSF from over 200 patients with a wide spectrum of neurological diseases were analyzed by 2D electrophoresis in addition to agarose electrophoresis. After a 2D run several hundred fractions of proteins were obtained. By perfecting this method the authors have succeeded in demonstrating oligoclonal zone patterns not previously described in the light chain region of the immunoglobulins in CSF samples. All patients with definite diagnosis of multiple sclerosis (MS), and also other diseases such as neurolues, myelitis, herpes zoster, etc. showed particular oligoclonal zone patterns. This experience indicates that the detection of oligoclonal zones in the region of the Ig-light chain is of major diagnostic significance. Moreover, the region of the so-called "CSF-specific" proteins deserves special attention.