Validation of estimated muscle fiber conduction velocity with the normalized peak-averaging technique.

To estimate muscle fiber conduction velocity (MFCV), the normalized peak-averaging technique was developed along with complementary software and reported in a previous study. Electromyograms were recorded from the vicinity of end-plate zones (EMG data 1) and a site farther from the end-plates and distal tendons (EMG data 2) of biceps brachii muscles using surface array electrodes during 50% maximal voluntary isometric contractions in ten healthy volunteers. The MFCV values obtained with normalized peak-averaging technique were compared to those obtained with the cross-correlation technique and examined by computer simulation of the MFCV distribution. The MFCV values obtained with normalized peak-averaging technique highly correlated with those obtained with cross-correlation technique in both EMG data 1 and EMG data 2. It was also confirmed that MFCV values obtained from EMG data 1 were distributed much more widely than those obtained from EMG data 2. These results show the clinical usefulness of normalized peak-averaging technique for the detailed assessment of MFCV.     

Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.     

A gel-sandwich technique for the qualitative and quantitative determination of dehydrogenases in the enzyme histochemistry. I. Development of the new methods on the example of LDH (E.C. 1.1.1.27).

A gel-sandwich technique for the histochemical demonstration of dehydrogenase is introduced with LDH set up as an example. Especially suitable, of the gels examined, for this technique is 1.5% W/V agar-agar low gel strength. In it several reaction ingredients for the histochemical reaction are dissolved. Considering LDH the following gel composition showed good results: 1.5% W/V agar-agar low gel strength, 5 mM TNBT in 150 microliter DMF, 120 mM L-lactate, 3--5 mM NAD+, 10 mM amytal, 22,4--32 X 10(-5) M Meldola Blue, 160 mM soldium phosphate buffer pH 7.6 (total solution of 1 ml). After the solidification of the gel, gel-bars were frozen with CO2-snow. The 40--80 micrometer thick gel slices were gained in the cryostat. Of the three different arrangement possibilities of the gel slices and the tissue-sections a sandwich arrangement (cover-gel slice--tissue section--ground-gel slice) produced the best results. The enzyme reaction is started by thawing of the gel slices (together with the tissue sections) and by putting them between the hotplate and the evaporator-head-piece, especially developed for this technique. The gel slices also remain in combination with the tissue sections after the reaction. The influence of the gel in combination with the electron carrier Meldola Blue on the spontaneous reduction rates of ditetrazolium salts in day light, were examined as well as the diffusion rates of TNBT and NADH out of gel slices and the influence of DMF and DMSO on the LDH activity. This technique prevents both, the loss of enzymes and the loss of reduction equivalents. There are given presuppositions for qualitative and quantitative histochemical investigations as well. The advantages of the new gel technique are discussed.     

Nuclear proteins. VI. Fractionation of chromosomal non-histone proteins using hydrophobic chromatography.

Mouse liver non-histone proteins, isolated by hydroxyapatite chromatography, were fractionated by hydrophobic chromatography using omega-amino-decyl-agarose omega-amino butyl-agarose, decyl-agarose, butyl-agarose, phenyl-Sepharose, and CPAD-Sepharose. Two column loading techniques were used. In the 0.35 M NaCl technique, the proteins were dialized into 0.35 M NaCl, applied to the column and initially eluted with 0.35 M NaCl. In the 40% (NH4)2SO4 technique, the non-histone proteins were mixed with the hydrophobic agarose, dialized against 40% (NH4)2SO4, and initially eluted with 40% (NH4)2SO4. In both cases the columns were subsequently eluted with 10 mM Tris-HCl, pH 7.5, 0.35, 1.0 and 5.0 M LiBr, and finally with 1% sodium dodecyl sulfate. The 0.35 M NaCl technique, using decyl-agarose and phenyl-Sepharose, resulted in a single step marked enrichment of the major hnRNA proteins (1 M LiBr fraction). The 40% (NH4)2SO4 technique resulted in a single step isolation of a pair of 15-20 000 dalton polypeptides.     

A new technique to study the mechanical properties of collagen lattices.

A new technique to study the mechanical properties of collagen lattices or dermal equivalents is described. With the help of the load-extension curve obtained with a uniaxial traction apparatus, a stiffness modulus varying from 0.06 to 1 MPa is calculated from the studied parameters (duration of culture, collagen and fibroblast concentration). The technique is presented as a new tool for testing the effect of pharmacological agents on the mechanical properties of the skin.     

Purification of the parasporal crystals ofBacillus thuringiensis subsp.israelensis and development of a novel bioassay technique

Summary A spore-free parasporal crystal suspension was prepared fromBacillus thuringiensis subsp.israelensis by an aqueous biphasic separation technique and the waste fractions quantified with regard to spore numbers and insecticidal potencies. The technique proved efficient in selectively removing spores from the spore-crystal mixture. The final crystal suspension was used to develop a novel bioassay system which allowed rapid determination of crystal effectiveness in 1/8 to 1/6 of the time required in the conventional mortality versus concentration bioassay.     

Studies on the pituitary "Fettstoffwechselhormon". VIII. Radioimmunoassay of the lipolytic factor P-LF II D.

A radioimmunoassay of the lipolytic peptide P-LF II D from porcine pituitaries is described. The assay is performed with 125-iodine labeled P-LF II D and with antisera either from guinea pigs or from rabbits. Bound antigen is separated from the free by double antibody technique. No cross reaction is observed with gamma lipotropin, peptide B, secretin, glucagon, isoproterenol. Due to contamination P-LF II C, beta lipotropin and human growth hormone displace the tracer when added at large doses. Complete cross reaction is observed between porcine 1-39 ACTH and P-LF II D. Specificity of this reaction is demonstrated by the increase of cross reaction, when ACTH fragments of increasing length of the polypeptide chain are used (1-23 ACTH, 1-24 ACTH and 1-28 ACTH).     

Studies on closure of the ductus arteriosus. X. In vivo effect of prostaglandin.

The effect of prostaglandins F2alpha, E1 and of 7-oxa-13-prostynoic acid on the newborn rabbit ductus can be studied using the whole-body freezing technique. PGF2alpha and PGE1 were able to re-open the closing ductus arteriosus in adequately oxygenated animals. PGF2alpha administration was accompanied by a strong physical reaction in the rat but less in the rabbit. PGE1 had sedative effects in both animals. A prostaglandin antagonist, 7-oxa-13-prostynoic acid had no effect on normal ductal closure nor did it counteract the effects of PGF2alpha and PGE1. The role of prostaglandins in homeostasis during the fetal and newborn period may be to modify ductal tone.     

Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping

The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2-nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission.     

Reducing the overlap problem in the proton NMR spectra of oligosaccharides by application of pseudo-four-dimensional homonuclear HOHAHA-HOHAHA-COSY.

A new homonuclear NMR experiment is described for the assignment of the proton NMR spectra of oligosaccharides, namely HOHAHA-HOHAHA-COSY (where HOHAHA is homonuclear Hartmann-Hahn spectroscopy and COSY is correlated spectroscopy). While this technique may formally be thought of as a four-dimensional NMR experiment, by use of selective pulses it is demonstrated that the analogous pseudo-four-dimensional experiment is a valuable improvement over conventional three-dimensional HOHAHA-COSY, in that the degree of resonance overlap is markedly reduced by the dispersion of resonances into a fourth effective dimension. The technique is demonstrated by application to the biantennary nonasaccharide Gal beta 1-4-GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man-beta 1-4GlcNAc beta 1-4GlcNAc.     

An experimental method for measuring force on the spinal facet joint: description and application of the method.

A technique is described for measuring load magnitude and resultant load contact location in the facet joint in response to applied loads and moments, and the technique applied to the canine lumbar spine motion segment. Due to the cantilever beam geometry of the cranial articular process, facet joint loads result in surface strains on the lateral aspect of the cranial articular process. Strains were quantified by four strain gages cemented to the bony surface of the process. Strain measured at any one gage depended on the loading site on the articular surface of the caudal facet and on the magnitude of the facet load. Determination of facet loads during in vitro motion segment testing required calibration of the strains to known loads of various magnitudes applied to multiple sites on the caudal facet. The technique is described in detail, including placement of the strain gages. There is good repeatability of strains to applied facet loads and the strains appear independent of load distribution area. Error in the technique depends on the location of the applied facet loads, but is only significant in nonphysiologic locations. The technique was validated by two independent methods in axial torsion. Application of the technique to five in vitro canine L2-3 motion segments testing resulted in facet loads (in newtons, N) of 74+ / -23 N (mean + / -STD) in 2 newton-meter, Nm, extension, to unloaded in flexion. Lateral bending resulted in loads in the right facet of 40+ / -32 N for 1 Nm right lateral bending and 54+ / -29 N for 1 Nm left lateral bending. 4 Nm Torsion with and without 100 N axial compression resulted in facet loads of 92+ / -27 N and 69+ / -19 N, respectively. The technique is applicable to dynamic and in vivo studies.     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. I. The radioimmunosorbent assay

A radioimmunosorbent technique is described which is capable of independently detecting both isozymes of carbonic anhydrase, CA I and CA II, in concentrations as low as 1 ng/ml. The technique is used to quantitate the different electrophoretic variants of red cell CA I as well as levels of CA II in the pig-tailed macaque, Macaca nemestrina.Supported by U.S. Public Health Service research grant GM-15419.U.S. Public Health Service Predoctoral Trainee (GM-71-14).     

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.     

Cloning the laboratory mouse.

A brief account is given of early attempts to clone mammals (mice) by transferring cells (nuclei) of preimplantation embryos into enucleated oocytes, zygotes or blastomeres of two–cell embryos. This is followed by a brief review of recent successes using adult somatic cells: mammary gland cells for sheep, muscle cells for cattle and cumulus cells for mice. We have developed a technique for cloning the laboratory mouse by transferring cumulus cell nuclei into enucleated oocytes. With this technique, we have produced a population of over 80 cloned animals, and have carried the process over four generations. Development and fertility of these appear normal. However, the yield is very low; only approximately 1*b/ of injected oocytes are carried to term. The challenge is now to understand the reason for this high loss. Is it a problem of technique, genomic reprogramming, somatic mutation, imprinting or incompatible cell cycle phases?     

Preparation procedures of proteins and RNA influence the total reconstitution of 50S subunits from E. coli ribosomes.

A two-step procedure has been described for the total reconstitution of 50S ribosomal subunit from E. coli. RNA and proteins are mixed with stoichiometry of 1:1.2 and incubated at 44 degrees C in 4.0 mM Mg2+ followed by a second incubation at 50 degrees C in 20 mM Mg2+ (Dohme and Nierhaus, J. Mol. Biol. 107, 585 (1976)). A modified method recently reported makes use of an altered preparation technique for the RNA and proteins and requires an RNA to protein stoichiometry of 1:2.5 and 7.5 mM Mg2+ in the first incubation (Amils et al., Nucl. Acid Res. 5, 2455 (1978)). The latter requirements are not compatible with the findings obtained with the first procedure. A comparison of the various RNA and protein fractions from the different groups revealed that the Mg2+ dependence of reconstitution is a function of the RNA preparation, whereas the stoichiometry depends upon the technique used for isolation of the protein fraction. The different RNA preparations were compared in the electron microscope.     

Occlusion pressures vs. micropipette pressures in the pulmonary circulation

Because of the discrepancies between the arterial and venous occlusion technique and the micropuncture technique in estimating pulmonary capillary pressure gradient, we compared measurements made with the two techniques in the same preparations (isolated left lower lobe of dog lung). In addition, we also obtained direct and reliable measurements of pressures in 0.9-mm arteries and veins using a retrograde catheterization technique, as well as a microvascular pressure made with the double-occlusion technique. The following conclusions were made from dog lobes perfused with autologous blood at normal flow rate of 500-600 ml/min and pressure gradient of 12 mmHg. 1) The double-occlusion technique measures pressure in the capillaries, 2) a small pressure gradient (0.5 mmHg) exists between 30- to 50-micron arteries and veins, 3) a large pressure gradient occurs in arteries and veins greater than 0.9 mm, 4) the arterial and venous occlusion techniques measure pressures in vessels that are less than 900 microns diam but greater than 50 microns, very likely close to 100 microns, 5) serotonin constricts arteries (larger and smaller than 0.9 mm) whereas histamine constricts veins (larger and smaller than 0.9 mm). Thus three different techniques (small retrograde catheter, arterial and venous occlusion, and micropuncture) show consistent results, confirming the presence of significant resistance in large arteries and veins with minimal resistance in the microcirculation.     

Development of a surface adhesion immunofluorescent technique for the rapid detection of Salmonella spp. from meat and poultry

A rapid method based on bacterial adhesion was developed for the detection of Salmonella in an enriched meat system. Minced beef samples inoculated with Salm. enteritidis (10 cfu g-1) were incubated overnight (18 h) at 37 degrees C in buffered peptone water. Salmonella enteritidis cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane attached to a glass slide. The organisms attached to this polycarbonate membrane were subsequently visualized using immunofluorescent microscopy. The technique had a detection level of log10 3.5 Salmonella ml-1. The surface adhesion immunofluorescent technique correlated well with Salmonella plate counts (r2 = 0.99). Application of the rapid method to retail beef and poultry samples (n = 100) confirmed the correlation between this technique and traditional microbiological procedures. Thirty-one retail samples were reported positive for Salmonella species. No false positives or negatives were recorded for the rapid method.     

Dissecting the chemistry of nicotinic receptor-ligand interactions with infrared difference spectroscopy.

The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.     

Salmonella mutagenicity of three complex mixtures assayed with the microsuspension technique. A WHO/IPCS/CSCM study.

In a collaborative study on complex mixtures, three complex mixtures and two pure compounds were assayed with the Salmonella microsuspension technique. The two pure compounds were benzo[a]pyrene (BaP) and 1-nitropyrene (1-NP). The three complex mixtures were standard reference materials (SRMs) from the U.S. National Institute of Standards and Technology, SRM 1649, SRM 1650 and SRM 1597. The two samples SRM 1649, an urban dust particulate matter, and SRM 1650, a diesel particulate matter, were sonicated with dichloromethane. Sample SRM 1597 was an extract of a coal tar sample with a complex mixture of polycyclic aromatic hydrocarbons. The microsuspension assay was performed with Salmonella strains TA98 and TA100 according to Kado et al. (1983) with minor modifications (L?froth et al., 1988). The results showed that the microsuspension technique is a more sensitive assay than the plate incorporation method. Depending on sample, strain and metabolic condition the mutagenic responses were 3-37 times higher in the microsuspension assay than in the conventional plate incorporation assay. The microsuspension method is thus useful for environmental samples which are often available in only small amounts.     

~1H-NMR用于肺炎链球菌荚膜多糖质量控制的尝试

纯化的6B、18C血清型肺炎链球菌荚膜多糖用生化试验和免疫学试验检测分析后再用一维氢谱核磁共振波谱(1H-NMR)法分析。生化检测其相应多糖的主要化学基团含量是否合格,免疫学检测旨在了解多糖纯化工艺是否影响了多糖的抗原活性,并间接佐证纯化多糖的生化特性是否正确。在此基础上进行1H-NMR分析,可以对纯化多糖的特性有进一步的了解。结果表明,常规的生化检测试验和免疫学检测试验并联合应用1H-NMR分析法后可更好地控制纯化肺炎链球菌荚膜多糖的质量。