A new method for detecting neural interconnectivity

We propose a class of counting process models for analyzing firing times of an ensemble of neurons. We allow the counting process intensities to be unspecified, unknown functions of the times passed since the most recent firings. Under this assumption we derive a class of statistics with their respective thresholds as well as graphical methods for detecting neural connectivity. We introduce a model under which detection is shown to be certain for long series of observations. We suggest ways to classify interactions as inhibition or excitation and to estimate their strengths. The power of the proposed methods is compared by simulating observations from artificial networks. By analyzing empirically obtained series we obtain results which are consistent with those obtained from cross-correlation-based methods but in addition obtain new insights on further aspects of the interactions. Received: 7 February 1996 / Accepted in revised form: 5 March 1997     

A method of detecting demyelinated nerve tissue in experimental allergic encephalomyelitis

A method of rapid pathomorphological diagnosis of demyelinization with a simultaneous detection of cell elements in the nervous tissue has been described. The method tested on the model of experimental allergic encephalomyelitis (EAE) is a modified method of Marchi with subsequent staining of histological sections with toluidine blue. Due to a shorter period of nervous tissue exposure to potassium bichromate and osmium acid solutions (from 6-8 weeks to 5 days) the cells preserve their ability to uptake the dye and the endurance of histological sections increases. Their thickness (2-3 microns) makes it possible to examine them in immersion magnification of the light microscope. Using the above method, we succeeded in revealing hematogenic and glial elements in demyelinating foci in the central nervous system of animals with EAE. The method described can be used for studying pathomorphology of multiple sclerosis and other demyelinating diseases in humans and their experimental models, as well as for the express diagnosis of demyelinization in pathoanatomical practice.     

A simple colorimetric method for detecting cell viability in cultures of eukaryotic microorganisms

The dye methylene blue can be taken up by dead or severely damaged cells, but not by living cells. Based on this fact, a method was devised which permits quantitative determinations of injured cells in populations of microorganisms such asSaccharomyces cerevisiae, Rhodotorula glutinis, andEuglena gracilis. The percentage of damaged cells was determined by measuring, at 664 nm, the optical density of cell suspensions pretreated with 0.15 mM methylene blue for 6 min, a condition that does not affect cell integrity as determined by oxygen consumption and release of potassium ions. This technique is faster and simpler than the classical dye-exclusion and plate-counting methods.     

A new method for detecting endocytosed proteins.

A new reagent, DPSgt, is described which has been designed to label cell surface proteins at 0 degree C. The reagent is easily made; it is water soluble and contains a reactive impermeant ester at one end, a tyrosine which can be radioiodinated at the other, and a disulphide in-between. The label can be removed from cells by cleaving the disulphide linkage in it with glutathione at 0 degree C. When cells are warmed to 37 degrees C between labelling and reduction, labelled proteins which are endocytosed acquire resistance to reduction. This provides a simple way of measuring the endocytosis of surface proteins. The intracellular pools of transferrin and LDL receptors in K562 cells and fibroblasts have been estimated. The results indicate that intracellular receptors are in non-reducing compartments, and that uptake of average cell surface (by non-coated pit processes) in K562 cells is small.     

A new method for detecting anaerobic threshold by gas exchange

Excess CO2 is generated when lactate is increased during exercise because its [H+] is buffered primarily by HCO-3 (22 ml for each meq of lactic acid). We developed a method to detect the anaerobic threshold (AT), using computerized regression analysis of the slopes of the CO2 uptake (VCO2) vs. O2 uptake (VO2) plot, which detects the beginning of the excess CO2 output generated from the buffering of [H+], termed the V-slope method. From incremental exercise tests on 10 subjects, the point of excess CO2 output (AT) predicted closely the lactate and HCO-3 thresholds. The mean gas exchange AT was found to correspond to a small increment of lactate above the mathematically defined lactate threshold [0.50 +/- 0.34 (SD) meq/l] and not to differ significantly from the estimated HCO-3 threshold. The mean VO2 at AT computed by the V-slope analysis did not differ significantly from the mean value determined by a panel of six experienced reviewers using traditional visual methods, but the AT could be more reliably determined by the V-slope method. The respiratory compensation point, detected separately by examining the minute ventilation vs. VCO2 plot, was consistently higher than the AT (2.51 +/- 0.42 vs. 1.83 +/- 0.30 l/min of VO2). This method for determining the AT has significant advantages over others that depend on regular breathing pattern and respiratory chemosensitivity.     

A new method for detecting single nucleotide polymorphism using GFP-display

The single nucleotide polymorphism (SNP) of aldehyde dehydrogenase-2 (ALDH2) codon 487, GAA (Glu) or AAA (Lys), was examined using green fluorescent protein (GFP)-display, an electrophoretic detection method for single amino acid changes. Although no shift in migration between the GFP-ALDH (Glu487) and GFP-ALDH (Lys487) fusion proteins was observed on SDS/urea gel, the two migrated to different positions when tagged with Asp. The SNP analysis was performed with GFP-ALDH-Asp3, and GFP-ALDH-Asp3 constructed from donors having the codon GAA/GAA, GAA/AAA or AAA/AAA was detected as different patterns as expected. GFP-display is potentially a unique method in SNP analysis, which does not require any special equipment or chemicals.     

A new method for obtaining bacteria-free cultures of blue-green algae

Blue-green algae are enriched and subcultured in a nitrogen-free medium. Portions of these cultures are kept at 47–48 C for 15, 30, 45 and more min. If akinetes are present and spore-forming bacteria absent — as is often the case — bacteria-free cultures of the algae are obtained.     

A new method for detecting sites of 2'-O-methylation in RNA molecules.

2'-O-methylation of eukaryotic ribosomal RNAs occurs in the cell nucleoli. At least 100 modification sites that are highly conserved among vertebrate rRNAs have been mapped. However, in part because of the insensitivity of current approaches, there are 2'-O-methylated sites that remain unidentified. We have developed an extremely sensitive method for detecting 2'-O-methylated residues that are predicted within a long RNA molecule. Utilizing RNase H cleavage directed by a 2'-O-methyl RNA-DNA chimeric oligonucleotide, this method has allowed identification of two methylated nucleotides, G1448 in Xenopus 18S rRNA and A394 in Xenopus 28S rRNA. The latter (A394 in 28S) had not been detected before. We have confirmed that the methylation at G1448 in 18S is dependent upon Xenopus U25 snoRNA and have demonstrated that the methylation at A394 in 28S requires U26 snoRNA. One advantage of this technique is that it can examine specific rRNA and precursor molecules. We show that about 30% of the 40S pre-rRNA has been methylated at these two sites and their methylation is complete at the stage of 20S (immediate precursor to 18S) and 32S (immediate precursor to 28S). We also show that methylation at these two sites is not essential for rRNA transport from the nucleus to the cytoplasm.     

Frozen protein arrays: a new method for arraying and detecting recombinant and native tissue proteins

DNA microarrays are powerful tools for high throughput analysis of gene expression; however, they do not measure protein expression. Current methods for producing protein arrays require sophisticated equipment or extensive protein modification. We developed a low overhead, customizable assay platform called frozen protein arrays that can detect native proteins in protein lysates. Frozen protein arrays were formed from a block of frozen histologic embedding compound containing an array of wells. The wells were filled with samples, which freeze and bond to the block. Cryosections were cut and transferred to nitrocellulose-coated slides. The reproducibility, linearity, and sensitivity was confirmed using frozen protein arrays filled with prostate specific antigen. Frozen protein arrays could detect native tissue proteins. The alpha1 subunit of NaK-ATPase was detected in rat kidneys with a coefficient of variation of 4.3-6.6%. Frozen protein array analysis indicated that the protein abundance decreased by 48.7% following renal ischemia, similar to the 40% decrease by Western blotting. We conclude that frozen protein arrays are a low cost, moderate size platform for arraying samples including protein lysates. Production of many identical frozen protein arrays is easy, inexpensive, and requires only small sample volumes. The method is gentle on proteins as they remain frozen during production.