[Prospects of using the electrophoresis technique for identification of the taxonomic status of earthworms (Lumbricidae)].

The polyacrylamide gel electrophoresis technique was used for comparison of common protein of body wall tissues in five species of Lumbricidae (Eisenia nordenskioldi, E.fetida, Lumbricus rubellus, Aporrectodea caliginosa, A. longa). The statistic processing of indexes of electrophoretic similarity revealed three levels of similarity: intraspecies, interspecies, and intergenus. It was discovered that the similarity of E. nordenskioldi and E.fetida proteins corresponded to the intergenus level. The use of this method for determination of the earthworm's taxonomic status is discussed.

[Carbon dioxide of air inhibits the formation of silver nanoparticles initiated by proteins in polyacrylamide gel and in solution].

It was shown that the detection of proteins in polyacrylamide gel by silver is inhibited by contact with air of the ammonia complex with silver ions used at the first stage of detection. It was proved by experiments on the reduction of silver by ethanolamine from a complex with ethanolamine and by formaldehyde from a complex with ammonia that the formation of silver nanoparticles initiated by proteins is inhibited by air carbon dioxide. The participation of carbon dioxide in this process is discussed. It was found that even the breathing of an experimenter can induce variations in carbon dioxide concentration sufficient to adversely affect the reproducibility of the silver staining techniques. It was concluded that, for stable staining of proteins by silver in polyacrylamide gel, it is necessary to maintain a low concentration of carbon dioxide in air over the detection solutions.

[Specific aspects of detection of peroxidase isozymes and their genetic control in barley].

Identical specimens were separated by electrophoresis in two gels to detect and fix peroxidase isozymes. Both gels were stained by Coomassie brilliant blue for detecting proteins. One gel was previously incubated for detecting peroxidase activity. The differences in electrophoretic patterns between the gels indicate the zones of peroxidase activity. It has been shown that locus Prx 6H, controlling a low-mobility grain peroxidase (PRX 6H), is localized to barley chromosome 6. Two loci, Alb 4H and Alb 7H, controlling the biosyntheses of water-soluble proteins of barley endosperm, were localized to chromosomes 4 and 7. It has been demonstrated that barley culture is polymorphic at multiple molecular forms of peroxidase.

Use of cationic disc electrophoresis near neutral pH in the evaluation of trace proteins in human plasma.

Cationic discontinuous electrophoresis can be carried out at pH 6.0 With excellent resolution in urea-containing acrylamide gels with potassium as the leading ion, 3-picoline as the trailing ion, and cacodylic acid as the buffer. This analytic technique has consistently demonstrated trace protein abnormalities in plasma or serum from febrile patients. It has made possible the detection of three protein bands that were obscured in similar electrophoretic assays at pH 3.8 A rough parallelism is found in the plasma (or serum) content of the five protein bands regardless of the apparent clinical cause of the febrile state.

Serum tyrosinase in malignant disease, its activity, and the electrophoretic patterns of the enzyme as carried by immunoglobulins'.

Serum tyrosinase activity in many persons with metastatic diseases was found to be significantly higher than activity in normal persons. The highest activity was observed in melanoma and breast carcinoma. The electrophoretic patterns of serum tyrosinase, resolved by electrophoresis of a serum tyrosinase fraction followed by incubation of the gel sample with L-dopa, and represented as sets of RF's of melanin bands, were characteristically different in melanoma, breast carcinoma, and certain other diseases. The RF's of melanin and protein bands in the serum enzyme preparations from melanoma patients were concisely defined. Further, some potent serum fractions inhibiting tyrosinase melanogenic activity have been obtained, and the presence of tyrosinase inhibitors in the serum enzyme preparation has also been demonstrated. More detailed exploration of these serum tyrosinase parameters may provide more specific and sensitive detection for certain malignant diseases.

Electrophoresis: the march of pennies, the march of dimes.

The present review encompasses ca. 65 years of history of developments in electrokinetic separations, taking as a starting point the year 1937, i.e. the official launching of Tiselius' moving boundary electrophoresis (MBE). The 1950s have been particularly rich in introducing novel methodologies in zone electrophoresis (ZE), thus bringing about the decline of MBE. Among them of extraordinary importance was the development of electrophoresis on agar gels coupled to immuno-diffusion at right angles, which brought a big revolution not only in biochemistry but also in clinical chemistry. Also the by now forgotten paper electrophoresis was a landmark in separation science, in that it implemented, in its "fingerprinting" version, the first genuine two-dimensional (2D) map, coupling orthogonally a charge to a hydrophobic scale separation, while permitting for the first time the detection of spot mutations, i.e. single amino acid replacements in a polypeptide chain, that paved the way to modern genetic analysis. Equally important was the introduction of starch-block electrophoresis, that brought about the notion of sieving and the first discontinuous buffers, refined, in the 1960s, by Ornstein and Davies with their classical papers combining multiphasic buffer systems to polyacrylamide gels, that went down to history as disc-electrophoresis. The 1960s also contributed with two fundamental techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that permitted to discriminate proteins solely on the basis of surface charge and molecular mass, respectively. The 1970s gave other fundamental contributions, such as isotachophoresis, the first example of a fully instrumental approach to electrophoresis, both in its analytical and preparative version (Tachophor and Tachofrac), 2D maps combining IEF to SDS-PAGE at right angles and silver staining techniques, that incremented sensitivity by 3 orders of magnitude. The 1980s generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

Characterization of dysfunctional factor VIII molecules.

Immunopurification and characterization of dysfunctional factor VIII-like molecules in CRM-positive and CRM-reduced hemophilia A permit correlation of structural changes with molecular defects. The technique described here is sufficiently sensitive to characterize the molecular mass and enzymatic fragments of the factor VIII chains in patients with as little VIII: Ag as 0.05 units/ml. Specific abnormalities have been identified in 5 of the first 24 samples tested. In each case, the mutation responsible for factor VIII dysfunction has been determined by sequencing a part of the abnormal gene. Mutations have been identified that abolish critical thrombin cleavage sites or which generate new N-glycosylation sites. The technique provides a useful approach to the study factor VIII structure-function relationships, and it has the potential to clarify further the molecular basis of factor VIII procoagulant activity.

Effect of stirring during fixation upon immunofluorescence. Results with distribution of albumin-producing cells in liver.

When the immunofluroscent study on the distribution and the incidence of albumin-producing hepatocytes in the rat liver was performed by the method of Sainte-Marie, the number of positive cells showed various values (10-60%). It was surmised that when the permeability of the fixative was delayed, albumin had flowed out from the cytoplasm of the unfixed hepatocytes. By the simple means of constant stirring of the fixative using a magnetic stirrer, we accomplished rapid fixation and achieved results in which positive cells attained 100%. On the other hand, the incidence of positive cells decreased markedly when rats were fed a protein-free diet.

Characteristic protein fractions of cerebrospinal fluid disc electrophoretic analysis.

The present work was undertaken to determine characteristic proteins of normal cerebrospinal fluid (CSF) by disc electrophoresis in polyacrylamide gel, and to evaluate their usefulness as indicators of blood-brain-CSF barrier disturbance. The technique has been applied to 1280 samples of unconcentrated CSF obtained from 27 reference subjects and 847 neurological patients, with a simultaneous analysis of 361 sera. The pre-albumin content (mean +/- S.D. as a percentage of total protein, 11.0 +/- 2.3%) was higher than formerly reported. One reason for this is that preliminary concentration was not necessary, and the second is related to the principle of protein resolution. The no. 5 protein band of the post-albumin group (3.9 +/- 1.1%) was characteristic, though it has not yet been identified. The no. 3 protein band of the post-transferrin group (5.2 +/- 1.7%) was highly specific to CSF; it was found to be closely related to transferrin, and may correspond to tau fraction obtained by other methods. Barrier dysfunction was easily recognizable by the appearance of polymers of haptoglobin 2-1 or 2-2, because only haptoglobin 1-1 was detected in normal CSF. The percentage of the main region of the G-zone (13.7 +/- 2.6%) was postulated as normal content of CSF immunoglobulins.

Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins.

Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.

Isotachophoresis of CSF proteins in gel tubes especially gammaglobulins. An analytical and preparative technique for high-separation of CSF proteins.

An isotachophoretic method using polyacrylamide gel (PAG-ITP) in a simple disc electrophoretic equipment with plastic tubes containing the gels, was elaborated and especially designed for studying the gammaglobulins in CSF and serum from control subjects and patients with neurological disorders, especially known or probable MS. The device and the ITP system used, including leading and terminating electrolytes and spacer substances, dividing the gammaglobulins in a reproducible way, are described. No cooling of the gel tubes was needed. The sample volumes varied between 5--500 microliters, and the separation time was 1.5--3.0 h. CSF from patients with verified or probable MS revealed characteristic, increased low-mobility gammaglobulin fractions. Using other ITP systems, such as other spacer compositions, the anodic proteins can also be studied in more detail. PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative procedures for biological material such as CSF, serum and extractions from nervous tissues.

Multimeric analysis of von Willebrand factor by vertical sodium dodecyl sulphate agarose gel electrophoresis, vacuum blotting technology and sensitive visualization by alkaline phosphatase anti-alkaline phosphatase complex.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

Evaluation of urinary proteins by sodium dodecyl sulphate polyacrylamide disc gel electrophoresis and molecular mass analysis.

Patterns of urinary proteins were examined on sodium dodecyl sulphate polyacrylamide gels and compared to those obtained using high voltage agarose electrophoresis. Both were found to be well adapted to routine laboratory analysis although the former had more sensitivity for early renal changes. Glomerular, mixed and tubular patterns were identifiable, and special precautions and pitfalls whilst interpreting the latter were pointed out. Restricted alpha 1 antitrypsin was shown in renal transplant proteinuria and its possible mechanisms were discussed. Molecular masses and frequencies of several discrete bands in heavy proteinurics were calculated, and attempts to identify them were made with reference to standard proteins.

Comparison of electrophoretic techniques for the analysis of human tear fluid proteins.

Five electrophoretic techniques were tested for application to the analysis of human tear proteins: disc-, SDS- and gradient polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. Protein patterns obtained from pooled normal tear fluid, tear fluid from keratoconjunctivitis sicca patients and from patients with chronic non-infectious conjunctivitis were compared. The four major components of human tear fluid were purified and their positions within the separation patterns were determined. The highest resolution and the clearest differences between the patterns of the three tear samples were obtained by SDS polyacrylamide gel electrophoresis.

Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan banded krait (Bungarus multicinctus) venom.

By ion exchange chromatography followed by gel filtration, a polypeptide toxin was purified from Formosan banded krait (Bungarus multicinctus) venom. The toxin had a molecular weight of 7000 +/- 300 and showed an amino acid composition characteristic of cardiotoxin from cobra venom. The i.p. LD50 value of the toxin was 2.5 (1.9-3.2) mg per kg mouse. Pharmacological studies showed that the toxin (10 micrograms/ml) induced contracture in chick and mouse skeletal muscles, depolarized the cell membrane of the mouse diaphragm, arrested the contraction of spontaneously beating atria and the electrically driven ventricle strip of the rat, and caused direct hemolysis of guinea-pig erythrocytes. From these chemical and pharmacological characterizations it was concluded that this toxin has characteristics similar to those of cobra venom cardiotoxins.

Electrophoretic pattern of serum glycoproteins on polyacrylamide disc gel in patients with breast cancer.

Numerous investigators have identified, isolated and characterized serum glycoproteins that are claimed to be specifically associated with malignancy. We have carried out serum glycoprotein electrophoresis on polyacrylamide disc gel in 53 breast cancer patients, at diagnosis as well as during and after therapy. Follow-up samples were divided into complete responders (CR) (n = 138) and nonresponders (NR) (n = 44). Glycoprotein electrophoresis showed multiple bands for each sample which were categorized into four groups: albumin, alpha, beta and gamma. The results revealed a decreasing number of CR and increasing number of NR with elevated (as compared to pretreatment levels) albumin fraction glycoproteins. Gamma region glycoproteins showed the reverse pattern to that of albumin region glycoproteins. The alpha and beta region glycoproteins revealed an increasing number of CR having higher values with increase in follow-up duration. In comparison with their pretreatment values CR showed significantly increased (Paired "t" test) values of albumin, alpha and beta region glycoproteins (p < 0.01, p < 0.001 and p < 0.001, respectively) and decreased gamma region glycoproteins (p < 0.001). The albumin, alpha, beta and gamma region glycoprotein levels were comparable between NR and untreated cancer patients. The variations in albumin, alpha, beta and gamma region glycoproteins correlate with treatment response, which might be useful in the treatment monitoring, and prediction of recurrence in breast cancer patients.

Microdisc-electrophoretic study of deoxyribonucleases in cow snout epidermis.

Acid and neutral deoxyribonucleases (DNases) of the cow snout epidermis were investigated by the microdisc-electrophoresis of polyacrylamide gels containing highly polymerized DNA and by isoelectric focusing techniques. The nucleases were characterized with respect to their pH optimum. An acid DNase at pH 5.0 was detected as a single distinct band after the electrophoretic separation. After isoelectric focussing also, only one acid DNase activity with an isoelectric point (IP) of 6.2 was detectable. Neutral DNases at pH 7.4 were demonstrated as major and minor bands by their different electrophoretic mobilities. In the isoelectric focusing system also, two neutral DNases, a major one (IP, 4.6) and a minor one (IP, 6.4), were found. Characterization with respect to their histologic location showed acid and neutral DNases across the epidermal layers with the highest activities in the upper layers, where DNA concentration had been shown to be lowest. In correlation with their subcellular distribution, the highest activities of both acid and neutral DNase were found in the 105,00 X g supernatant of the subcellular fractions.

Automatic microdisc electrophoresis for urinary protein analysis: a comparison with standard analysis.

Molecular weight related urinary protein analysis is a very valuable diagnostic tool in modern nephrology. However, until recently urine had to be prepared for analysis by several procedures including concentration. Using a new micro-method it was demonstrated that the clinical significance of urinary disc-electrophoresis is much higher than with former systems. Thus, the use of microdisc electrophoresis (Phast system) is recommended for urinary protein analysis in the future.

Reduced red blood cell carbonic anhydrase activity in a patient with nephrolithiasis.

Low levels of red blood cell (RBC) carbonic anhydrase (CA) activity in hemoglobin-free hemolyzate (HFH) were found in 2 children with nephrolithiasis when compared to age-matched controls. The lowest levels were consistently found over a two year period in a 6 1/2 year old boy (U.P.) whose renal calculi contained uric acid, ammonia, calcium carbonate and oxalate. His RBC CA values ranged from 1.4-2.7 U/g Hb compared with levels of 3.9-5.3 U/g Hb in control subjects. Statistical comparisons of the mean values for U.P., 2.06 U/g Hb, and his age-matched control subjects, 4.46 U/g Hb, revealed a significant difference (P = less than 0.001). Similar reductions in RBC CA activity were found in his father--2.0 and 2.1 U/g Hb compared with 3.3 and 3.9 U/g Hb in adult controls. HFH CA activity was not decreased in the mother or sister. Polyacrylamide gel electrophoresis of HFH from 4 of the children and the father of U.P. was abnormal. However, this abnormal electrophoretic pattern could only be demonstrated when the gel was run for 120 minutes and not when it was run for 80 or 160 minutes. We have identified a patient and his father with low levels of RBC CA activity.

The comparative analysis of clinically normal and inflamed human gingiva. Study 1: Protein separation by anionic and cationic disc gel electrophoresis.

Homogenates were prepared from sections of both clinically normal and clinically inflamed human gingival tissue. After centrifugation, supernatants were quantitated for protein and subjected to anionic and cationic electrophoresis. Samples of gingival blood and venous blood were similarly subjected to electrophoresis. Significant protein pattern differences are reported for the anionic tissue separations, and for the cationic tissue and plasma separations.