Acid Probes Research Articles

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

The eye lens beta-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized beta-crystallin sequences (beta A2, beta A3/A1, beta A4, beta B1, beta B2, beta B3), isolated from a bovine lens cDNA library, to Southern blots on which EcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic beta B-crystallins gave hybridization signals in more species than the acidic beta A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for beta A2 was found in trout and lamprey, for beta A4 in trout, and for beta A3/A1 only in toad. The results demonstrate that the duplications leading to the beta-crystallin gene family occurred before or during the earliest stages of vertebrate evolution.

Characterization of drug binding sites on .ALPHA.1-acid glycoprotein.

The classification of drug binding sites on alpha 1-acid glycoprotein (AGP) was studied by displacement experiments using fluorescent probes. Basic drugs not only displaced basic probes strongly but also acidic probes as well. Acidic probes, on the other hand, were displaced by some acidic drugs such as phenylbutazone and sulfadimethoxine which had no effect on most of the basic probes. This contradiction suggests that the basic drugs do not completely share a binding site with the acidic drugs. The polarity of the basic drug binding site was higher than that of the acidic drug binding site. The negative charges were probably located in or near the former, different from the latter. The basic drug binding site was more sensitive to the conformational change of AGP. It seems that there are particular drug binding sites on the AGP molecule for acidic and basic drugs. However, all the displacement data do not fully support the possibility of two independent drug binding sites. Therefore, it is rather reasonable to consider that these sites are not completely separated but are significantly overlapped and influenced by each other. Accordingly, AGP seems to have one wide and flexible drug binding area.

Topological distribution of atropine in dipalmitoylphosphatidylcholine liposomes

The topography of atropine entrapped in sn-3-(dipalmitoyl)phosphatidylcholine (DPPC) liposomes was determined by the electron spin resonance (ESR) technique using 5-, 7-, and 16-nitroxy-stearic acid probes. The liposome preparations, with or without entrapped atropine, were passed through a Sepharose-4B column; the entrapment efficiency and lipid recovery were determined using [3H]-atropine and [14C]-DPPC as tracers. It was found that approximately 31 per cent of the added atropine was entrapped in the liposomes. The ESR results established that the temperature range of the gel-to-liquid crystalline transition of DPPC liposomes was not altered significantly by the encapsulation of atropine. The entrapped atropine also caused no significant change in the order of hydrocarbon chains of DPPC vesicles. These results were confirmed by data obtained from differential scanning calorimetry. On the basis of these experiments, it was concluded that atropine does not interact with the hydrophobic region of the lipid bilayers but is exclusively localized within the entrapped aqueous compartment of DPPC liposomes.

New Methods for Identification of Obligate Anaerobes

Anaerobes are identified in the clinical laboratory to provide a microbiologic diagnosis for the individual patient as well as information on pathogenicity and epidemiology. The level of identification required in each context should be discussed and agreement should be reached. New methods of identification available, or likely to become available, include nucleic acid and immunologic probes, protein electrophoresis, peptidoglycan analysis, detection of volatile metabolic products, and detection of enzymes. The last of these has been widely adopted for the production of commercially available identification kits. All the methods require critical assessment before they are introduced into routine service.

Sex Determination of Bloodstains by the Use of a Ribonucleic Acid (RNA) Probe

An asymmetric ribonucleic acid (RNA) probe, which represents a Y-chromosome specific nucleotide sequence, was applied to the sex origin of bloodstains by using a nonisotopic Southern hybridization method. The RNA probe was in vitro transcribed from the recombinant of the Y-chromosome specific deoxyribonucleic acid (DNA) sequence and transcription-vector Bluescript SK M13+. The use of this RNA probe can lower the detection limit of the nonisotopic DNA analyzing method to 1/8 of that reached by our previously reported method in which a DNA probe was used.

A Study of Sex Identification of Trace, Dried Bloodstains Using a Y-Chromosome-Specific Deoxyribonucleic Acid (DNA) Probe

A new method is discussed which examines trace, dried bloodstains by gel in situ hybridization using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe to determine the sex of the bloodstain for forensic medicine application. The complete DNA is transferred directly by electrophoresis onto the gel intact, bypassing the possibilities of impurities contaminating the sample and of DNA degradation. The method has proven accurate for small (2.5-mm-diameter) samples aged up to eight years and is quick, simple, and easily read.

Identification of Male Bloodstains by Dot Hybridization of Human Y Chromosome-Specific Deoxyribonucleic Acid (DNA) Probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.

A milling crowd model for local and long-range obstructed lateral diffusion. Mobility of excimeric probes in the membrane of intact erythrocytes

A new model for lateral diffusion, the milling crowd model (MC), is proposed and is used to derive the dependence of the monomeric and excimeric fluorescence yields of excimeric membrane probes on their concentration. According to the MC model, probes migrate by performing spatial exchanges with a randomly chosen nearest neighbor (lipid or probe). Only nearest neighbor probes, one of which is in the excited state, may form an excimer. The exchange frequency, and hence the local lateral diffusion coefficient, may then be determined from experiment with the aid of computer simulation of the excimer formation kinetics. The same model is also used to study the long-range lateral diffusion coefficient of probes in the presence of obstacles (e.g., membrane proteins). The dependence of the monomeric and excimeric fluorescence yields of 1-pyrene-dodecanoic acid probes on their concentration in the membranes of intact erythrocytes was measured and compared with the prediction of the MC model. The analysis yields an excimer formation rate for nearest neighbor molecules of approximately 1 X 10(7) s-1 and an exchange frequency of approximately greater than 2 X 10(7) s-1, corresponding to a local diffusion coefficient of greater than 3 X 10(-8) cm2 s-1. This value is several times larger than the long-range diffusion coefficient for a similar system measured in fluorescence photobleaching recovery experiments. The difference is explained by the fact that long-range diffusion is obstructed by dispersed membrane proteins and is therefore greatly reduced when compared to free diffusion. The dependence of the diffusion coefficient on the fractional area covered by obstacles and on their size is derived from MC simulations and is compared to those of other theories lateral diffusibility.

Differential Thermal Analysis as an Acidity Probe in Zeolite Catalysts

Abstract Differential thermal analysis is used as an acidity strength probe for a series of mordenite (zeolite) catalysts via determining the temperature at which presorbed pyridine completely desorbs from the catalyst. The findings obtained for metal(s) containing mordenite catalysts have been correlated with the Pearson's assumption concerning Lewis acids and bases and the Pauling's electronegativity of the metals contained and found compatible. The investigated catalysts can be arranged in their observed order of acidity strength as follows: HTI > Th-HM > Re-HM > Pt-HM > Pt-Th-HM > Pt-Re-HM.

Topological Reaction Rate Measurements Related to Scuffing

The contact area of the plate of a ball/plate (both consisting of hardened M-50 steel) sliding elastohydrodynamic contact run on trimethylolpropane triheptanoate with or without tricresylphosphate (TCP) additive was optically profiled periodically with a phase-locked interference microscope (PLIM), both before and after exposure to alcoholic hydrochloric acid. As scuffing was approached, the change of profile within the contact region changed much more rapidly by the acid probe and assumed a constant high value after scuffing. A new nonetching metallurgical phase was found in the scuff mark, which was apparently responsible for the high reactivity. Soaking the metal surfaces in the lubricant before running affected the subsequent reactivity. The microscopic profile changes (sensitivity ± 30 A in depth) involved primarily the small asperities of radius < 3 μm, while the larger ones were unaffected. Soaking the steel in TCP smoothed the fine structure of the surface profile, but increased the reactivity tow...

Spin-label studies of erythrocytes in myotonic dystrophy: no increase in membrane fluidity.

The basic defect in myotonic dystrophy is thought to involve muscle cell membranes. Butterfield and associates have recently presented electron spin resonance data that suggest increased fluidity of erythrocyte membranes in patients with myotonic dystrophy. We studied erythrocytes from 11 patients with myotonic dystrophy and 14 age-matched controls, using spin-labeled fatty acid and ester probes. Despite attempts to reproduce the previously reported experimental conditions exactly, we found no significant differences in the electron spin resonance spectra of erythrocytes from normal and myotonic dystrophy subjects. These findings do not provide evidence of increased erythrocyte membrane fluidity in myotonic dystrophy; they fail to support the concept of an intrinsic defect of the lipid membrane in this disorder.

Lipid-lipid and lipid-protein interactions as studied with a novel type of fluorescent fatty acid and phospholipid probes.

A novel fluorescent-labelled group of fatty acids and phospholipids has been applied to determine phase transitions in liposomes by fluorescence intensity and polarisation measurements. The chromophore of these amphiphilic lipids proved to be very suitable to demonstrate temperature-dependent lipid-lipid interactions. Liposomes from 1,2-dipalmitoyl-3-sn-glycero-phosphoethanolamine and from lipids isolated from membranes of E. coli K 1062 mutant grown on elaidic acid were used in these studies. These probes also made it possible to observe conformational changes in membrane proteins in isolated plasma membranes from this mutant. The changes in protein conformation were dependent on structural changes in the lipid phase.