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Background Although there are reports of robot-assisted ureteral reconstructions (RURs) with excellent safety and efficacy, the procedures remain technically challenging. In the robotic setting the surgeon must rely on visual cues in the absence of tactile feedback. Indocyanine green (ICG) is a dye that can be visualized under near-infrared fluorescence (NIRF). Objective To describe our novel technique, which utilizes intraureteral injection of ICG and subsequent visualization under NIRF to facilitate RUR, and report our outcomes after these procedures. Design, setting, and participants This is a retrospective review of 25 patients who underwent 26 RURs for various ureteral pathologies between June 2012 and October 2013. Surgical procedure After full disclosure, all patients consented to off-label use of ICG. A ureteral catheter and/or percutaneous nephrostomy tube were used to inject 10ml of ICG into the diseased ureter, above and below the stricture. Intraoperatively, NIRF was activated to assist in identification of the ureter and to localize the margins of ureteral strictures. Measurements Postoperatively, RURs were assessed for clinical success (absence of symptoms attributable to ureteral pathology) and radiological success (absence of a ureteral stricture on imaging). Results and limitations Our technique provided visual cues and aided in successful performance of 26 RURs in 25 patients. The procedures included ureterolysis ( n =4), pyeloplasty ( n =8), ureteroureterostomy ( n =9), and ureteroneocystostomy ( n =5). There were no perioperative complications attributable to ICG use. At a mean overall follow-up of 12 mo, all procedures were clinically and radiologically successful. This study is limited by the small sample size and short-term follow-up. Conclusions Intraureteral injection of ICG and subsequent visualization under NIRF facilitates RUR by aiding in rapid and accurate identification of the ureter, and precise localization of the proximal and distal ureteral stricture margins. In our experience, our technique is safe, easy to perform, and reproducible. Patient summary In this report, we describe a new technique to facilitate robot-assisted ureteral reconstructions using intraureteral injection of ICG and subsequent visualization under near-infrared fluorescence. More specifically, our technique allows for rapid and accurate identification of the ureter, and precise localization of ureteral strictures.

increased DNA fragmentation and clinical outcome from IMSI, although results are conflicting. The aim of this study was to compare pregnancy rate (PR) from standard intra-cytoplasmic sperm injection (ICSI) and IMSI, in couples who had failed previous ART attempts in whom the male partner had been demonstrated to have a raised DFI. METHODS: Data was collected prospectively between July 2011 and October 2012 at a fertility clinic. Couples who had undergone at least one previous failed attempt of ART at the clinic were advised DNA fragmentation testing using the TUNEL technique. DNA fragmentation was considered raised if the index (DFI) was above 30%. Couples were offered either standard ICSI or IMSI and PR, live birth rates (LBR) and miscarriage rates were compared. Sperm for injection with the IMSI technique were selected under 6600 x magnification based on the motile sperm organelle morphology examination (MSOME) as previously described by Bartoov et. al. RESULTS: 54 couples were included in the study analysis (ICSI, n1⁄420; IMSI n1⁄434). Mean age in women (ICSI, 38; IMSI, 37) (p1⁄40.14) and menwere similar in both groups (ICSI, 40; IMSI, 42) (p1⁄40.31). There was no statistical difference in themeanDFI in both coupleswho had ICSI and IMSI (ICSI, 44.3%; IMSI, 48.3%) (p1⁄40.34). Therewas no difference in the number of oocytes collected (ICSI, 9.0; IMSI, 12.5) (p1⁄40.06) or in the number of oocytes fertilized (ICSI, 5.1; IMSI 6.7) (p1⁄40.17). Two women who had ICSI had no fertilization of their oocytes on the day following egg collection and were excluded in the final analysis. Of women who had embryo transfer (n1⁄452), there was no difference in PR (ICSI, 55.6%; IMSI, 52.9%) (p1⁄40.56) or LBR (0.61)(ICSI, 50.0%; IMSI, 50.0%) between groups. Miscarriage rate was 5.6% in the ICSI group and 8.8% in the IMSI group. CONCLUSIONS: This study would suggest that IMSI may not improve the pregnancy outcome from ART in couples where men are found to have high DNA fragmentation.

Digitonin is widely used for extracting active neurotransmitter receptors from membranes. However, its low critical micellar concentration has made its removal from samples problematic. Here we report that digitonin can be efficiently removed (> 90%) from solution using Extracti-Gel D, a detergent-absorbing matrix. Active kappa 1 opioid receptors solubilized from brain survive Extracti-Gel D chromatography with a recovery of 50-55% and 25% dilution by added volume. The loss of receptor and the dilution, however, are compensated for to a large extent by the disinhibition of binding that results from the removal of digitonin. Extracti-Gel D chromatography had little or no effect on the apparent equilibrium dissociation constant for [3H]U-69,593 binding to the kappa 1 receptor. We conclude that Extracti-Gel D column chromatography is a simple, highly efficient and practical method for markedly reducing the concentration of digitonin in biological samples. Application of the procedure should allow characterization of digitonin-solubilized receptors with minimal complications from bound digitonin and extend the usefulness of digitonin to studies going beyond the initial stages of receptor purification.

Maternal consumption of ethanol produces a pattern of malformations, including nervous system abnormalities, in the developing fetus, a state called Fetal Alcohol Syndrome. We report the dose-dependent inhibition by ethanol of the growth of a glioma derived cell line, C6 cells; the effects occur at ethanol concentrations commonly encountered in the blood during human intoxication. The effects occur with different morphological subtypes of the cell line and do not occur when the cells are exposed to iso-osmolar concentrations of other chemicals. The results demonstrate that C6 cells are a model for the study of the effects of ethanol on nervous system cell growth.

A simple and highly sensitive assay for measuring total digitonin in biological samples is described. The assay is based on the ability of digitonin to hemolyze red blood cells. The precision and reproducibility of the assay was excellent with intra- and inter-assay variabilities of less than 1% and 6%, respectively. The assay was used to evaluate several potential methods for removing digitonin from biological samples (digitonin extracts from guinea pig brain membranes). Dialysis and G-25 Sephadex chromatography were ineffective. However, protein and digitonin can be effectively separated by ammonium sulfate precipitation followed by dialysis. The kappa 1 opioid receptor survived these procedures with no change in affinity for [3H]U-69,593. In conclusion, the hemolytic assay for digitonin appears to provide a practical means for determining detergent concentrations during receptor purification and characterization and for evaluating potential methods for detergent removal. Although an in depth analysis of the assay was carried out only for digitonin, CHAPS and deoxycholate also caused 50% hemolysis at concentrations well below those commonly used for receptor solubilization and, therefore, the general assay procedures might have applicability for measurement of these and perhaps other detergents used in receptor solubilization as well.

Alcohol metabolism in the human brain has been characterized as essentially nonoxidative in nature, with the esterification of ethanol with fatty acids via fatty acid ethyl ester synthase. This pathway of ethanol metabolism is related to end organ damage in the brain but the neural cell type expressing FAEES has not been identified. In this study human and rodent neuroblastoma and glioma cell lines are assayed for fatty acid ethyl ester synthase activity. Cells with neuronal properties demonstrated higher activity than glioma cell lines. We confirmed the presence of the mRNA for one type of synthase, fatty acid ethyl ester synthase-III in three neuronal cell lines - N1E115 cells, PC12 cells, and SK-N-MC cells. These results support the hypothesis that FAEES activity is expressed chiefly in cells with neuronal properties and suggest that non-oxidative ethanol metabolism is potentially related to the toxic effect of ethanol on the human brain.

The question of whether three acidic, water-soluble proteins (14-3-2, 14-3-3, and S-100, the first and last known to be brain-specific) are axonally transported was investigated in the rabbit visual system. The water-soluble proteins were obtained from individual optic nerves, combined optic tracts and lateral geniculate bodies, superior colliculi, and, in some instances, retinas at various times (1–56 days) after monocular injections of [3H]leucine. These proteins were separated by a two-step polyacrylamide gel electrophore-sis procedure that isolated 14-3-2, 14-3-3, and S-100 almost uncontaminated by other radioactivity. The isolated 14-3-2 and S-100 were demonstrated to be approx. 90% pure by a new method based on retarding the migration of these proteins by immunoadsorption during the first step of electrophoresis. An analysis of the radioactive labeling of the total soluble proteins (TSP) and the isolated acidic proteins revealed that: (1) S-100 was not axonally transported; (2) both 14-3-2 and 14-3-3 were part of one of the slow components of axonal transport (2-4 mm/day); (3) the radioactivity of 14-3-2 and 14-3-3 represented about 2.7% and 3.2%, respectively, of the radioactivity incorporated into the axonally transported TSP; (4) the ultimate distributions of the radioactively labeled 14-3-2 and 14-3-3 were the same (about 70% of each destined for the superior colliculus) and differed from that of the TSP; and (5) the rates of catabolism of the axonally transported 14-3-2 and 14-3-3 were slightly greater than that of the TSP, with half-lives for 14-3-2 and 14-3-3 estimated to be 11 and 10 days, respectively.

Synaptosomes were prepared from rat cortex by subjecting a washed crude mitochondrial pellet to centrifugation first on discontinuous Ficoll-isotonic sucrose gradients and then on discontinuous sucrose gradients. The synaptosome fraction, collected from the 7.5-14% Ficoll band (II), was further separated into two additional fractions, designated IIA and IIB, which bank at the 0.32-1.05 M and at the 1.05-1.6 M sucrose interfaces, respectively. Electron microscopic analysis showed that fraction IIB contained synaptosomes and extra terminal mitochondria and was essentially free of membrane fragments. Further characterization showed that IIB contained 69% of the protein and 83% of the lactic dehydrogenase activity of fraction II and had a specific activity of a 2',3'-cyclic nucleotide 3'-phosphohydrolase approximately 1% of that obtained with myelin. Fraction IIA had approximately 50% the specific activity of the 2',3'-cyclic nucleotide 3'-phosphohydrolase found in myelin. Synaptic plasma membranes were prepared by lysing fraction IIB in 1 mM sodium phosphate, 0.1 mM EDTA at pH 8.5 and subjecting this preparation to centrifugation on a discontinuous sucrose density gradient. Enzymatic analysis indicated that membranes banding at the 0.6-0.8 M sucrose interface had high specific activities of plasma membrane enzymes (e.g. acetylcholinesterase, ATPase, 5'-nucleotidase). The specific activity of the (Na+ + K+)-ATPase in the purified membrane preparation was 8-fold higher than that in the original homogenate. Specific activities of various marker enzymes indicated that the composition of these membrane preparations for the most part was synaptic plasma membranes, approximately 7% mitochondrial outer membranes and 3% a membrane containing 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. The polypeptide compositions of three possible contaminating membranes and of synaptic membranes were compared by electrophoresis in 6-20% gradient polyacrylamide gels in the presence of sodium dodecyl sulfate. Whereas mitochondrial and myelin membranes had distinct compositions, the compositions of the microsomal and synaptosomal plasma membranes were similar. Synaptic plasma membranes contained at least 27 polypeptides; the three major polypeptides had molecular weights of 103,000; 54,000; and 50,000. The major polypeptides of soluble synaptosomal proteins had molecular weights of 54,000 and 42,000.

The S-100 nervous system protein was purified from bovine and rat brains by a modification of the original procedure. The main modification consisted in substituting a step of calcium-dependent binding of S-100 to a phenyl-Sepharose column for the original step of chromatography on G-200 Sephadex. The proteins were pure as determined by SDS gel electrophoresis. HPLC on a reversed phase and on a size-separation column, and by immunological criteria. The bovine S-100 behaved as previously described, during calcium binding, by displaying a conformational change as evidenced by increase in native fluorescence.

Polyribosomes, carrying nascent polypeptide chains, were prepared from whole brain, cortex, and hindbrain-medullary white matter of young adult rats. In a homologous cell-free system, a brain-specific protein (S100 protein) was identified in the mixture of polypeptides released from the polyribosomes during incubation for 1 hr at 37°. De novo synthesis of the S100 protein was achieved in a reconstituted cerebral cell-free system containing polysome-derived mRNA and 40S + 60S subunits. The radioactively labeled S100 protein synthesized in vitro was identified by precipitation with antibody to S100 after addition of purified S100 as a carrier, and migration of the solubilized precipitate on acrylamide gels in the presence of sodium dodecyl sulfate. In vitro synthesis of the S100 protein did not occur in analogous cell-free systems derived from hepatic tissue or in a heterologous system containing liver polyribosomes and cerebral enzymes.

— —A soluble protein (S-100) which is unique to the nervous system was measured in rabbit tibial nerve at 0, 3, 7, 14, 21, and 28 days of degeneration. Amounts of S-100 in the degenerated peripheral segment of the transected nerve fell progressively during degeneration to 2 per cent of that measured in the corresponding portion of nerve taken from control rabbits 28 days postoperatively. Total soluble proteins increased 42 per cent during this time. Levels of S-100 and total soluble proteins remained unchanged in non-degenerated nerve segments from experimental and control rabbits. Correlations of amounts of S-100 measured in the study reported here with cellular changes demonstrated by other investigators to characterize Wallerian degeneration in peripheral nerve suggest that the S-100 protein is localized primarily in axons rather than in Schwann cells or myelin.

SUMMARY A method was developed for chromatography of rat liver soluble proteins on diethylaminoethyl cellulose, with a parabolic chloride gradient for elution. Protein, nucleic acid and 16 en- zyme activities were determined in the fractions, and each gave a consistent pattern of distribution in a number of preparations. Some of the enzyme activities, for example, lactic dehydro- genase, isomerase, and aldolase, were not adsorbed by the diethyl- aminoethyl cellulose at pH 8.0 and low ionic strength. Several of the enzymes gave multiple peaks of activity. With two of multiple peak enzymes, glucose 6-phosphate dehydrogenase and glutamic oxaloacetic transaminase, rechromatography of one of the peaks, under the same conditions, gave a single peak at the anticipated position. REFERENCES 1. SOBER, H. A., GUTTER, F. J., WYCOFF, M. M., AND PETERSON, E. A., .Z. Am. Chem. Sac., ‘78, 756 (1956). 2. SOBER

Free and membrane-bound polyribosomes were prepared from whole brain, cortex, and hindbrain-medullary white matter of young adult rats. In a homologous cell-free system, two brain-specific acidic proteins (S100 and 14-3-2) were identified in the soluble proteins released from the polyribosomes during incubation for 1 hr at 37°C. Both S100 and 14-3-2 proteins were found to be synthesized primarily by free polyribosomes. De novo synthesis of the 14-3-2 protein was achieved in a reconstituted cerebral cell-free system containing polysome-derived DNA-like RNA and 40S+60S subunits. The radioactively labeled S100 and 14-3-2 proteins were identified by immunoprecipitation with specific antisera and sodium dodecyl sulfate polyacrylamide gel electrophoresis. No difference was found in the synthesis of these brain-specific proteins when a hepatic supernatant enzyme fraction was used in the cell-free system containing cerebral polyribosomes. Furthermore, in vitro synthesis of S100 and 14-3-2 proteins did not occur with liver polysomes when a supernatant enzyme fraction from either liver or brain was used.

SummarySerum acid phosphatase represents a mixture of different molecular forms of the enzyme which can be separated and characterized. At least 2 major components can be separated by column chromatography with high recovery of the enzyme activity. The prostatic fraction of acid phosphatase appears, on the basis of its chromatographic behavior and its sensitivity to tartrate inhibition, to be localized in the second chromatographic peak.

SummaryThese results show that the above chromatographic procedure can be used to separate different protein extracts into a number of fractions and to investigate the distribution of various enzyme activities to pick out qualitative enzymic differences between malignant tumors and the normal tissues from which they arise.

A procedure was worked out for purification and identification of calcium-binding proteins from bovine brain using Ca2+-dependent, reversible binding to a hydrophobic support, phenyl-Sepharose, as the method of isolation. These proteins could be visualized during and after their separation by running them on non-denaturing polyacrylamide gels, blotting to Zeta-probe paper, and autoradiographing with 45Ca2+. About 24 polypeptides could be seen in this fraction on SDS (Laemmli) gels and about 8-10 native, Ca2+-binding proteins could be seen on non-denaturing gels and on blots of their 45Ca2+ autoradiographs. Some of these proteins could be purified further by chromatography on DEAE-Sephacel and still retain their 45Ca2+-binding activity.

The differentiated forms and functions of a specialized cell are expressed through the properties of its individual proteins, properties which are determined by their primary structure. Nervous system cells, having extremely specialized functions, are among the most highly differentiated of all types of cells. Therefore, it is important to know which proteins are specific to nervous system cells since these particular proteins would be related to specific functions within the nervous system, such as propagation of action potentials, synaptic transmission involving several chemical transmitters— each with its associated processes of synthesis, inactivation, release, and receptor activity, establishment of specific pathways and connections, action of supportive cells such as various types of glia, and many other functions.

The binding of Ca2+ to rat or bovine S-100 proteins, in the absence of ligands, showed a dissociation constant (in 60 mM K+) of 0.5 to 1.0 mM as measured by the effects of Ca2+ on binding of S-100 to phenyl-Sepharose, reactivity of sulfhydryl groups, and difference spectra for PHE, TYR, and TRP residues. Binding of the ligands, "Stainsall" and chlorpromazine lowered the dissociation constant of S-100 for Ca2+ by 2- to 10-fold as measured by the same parameters. The conformational change, in response to Ca2+ binding, probably occurs by exposure to solvent of the hydrophobic region of alpha and beta subunits of S-100 at residue positions 74-93.

Rubrophilin, a unique brain specific polypeptide, was purified to apparent homogeneity from microsomal fractions of bovine brains. The peptide stains pink with Coomassie Brilliant Blue R-250 (C.I. No. 42660) under specific conditions, has an apparent Mr of 53,000, and is acidic with an apparent pI of 4.9. The purification involves initial solubilization of delipidated microsomes in sodium dodecyl sulfate, followed by ammonium sulfate fractionation, reversed ammonium sulfate gradient elution from diatomaceous earth, gel filtration on polyacrylamide (Biogel P-200), gradient elution chromatography from hydroxylapatite, and reverse-phase chromatography from phenyl-Sepharose. A yield of about 5 mg of rubrophilin was obtained from 9 g of microsomal proteins. Amino acid analysis shows that rubrophilin contains only nine amino acids with residues/mol as follows: alanine (102), glutamic acid (97), lysine (65), proline (55), aspartic acid (48), glycine (44), serine (37), threonine (35), and valine (10). Cysteine, methionine, tryptophan, tyrosine, isoleucine, phenylalanine, histidine, and arginine could not be detected. Relative rubrophilin content of vertebrate brains was as follows: mammals greater than birds greater than reptiles greater than fishes. It is present in mouse retina and human neuroblastoma cell cultures but could not be detected in octopus optic lobe or in cultured C-6 rat glioma cells.

Summary Two aspects of the subunit structure of the bovine brain specific protein 14-3-2 have been examined. On the one hand, native 14-3-2 has been separated into two fractions by hydroxylapatite chromatography. One eluted at the same position when chromatographed on the same column, while the other redistributed into the same two fractions again. Amino acid analysis of these two forms of 14-3-2 gave results that were not significantly different under the condition of analysis. Furthermore, when each peak was subjected to cyanogen bromide cleavage, very similar elution profiles of the resultant fragments were obtained. The two pools also cross-reacted with antiserum to 14-3-2. Reaction of purified 14-3-2 with dimethylsuberimidate caused the formation of covalently bound protein units of 100,000 molecular weight when measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as opposed to the 50,000 minimal molecular weight normally detected. On the other hand, analysis of the soluble tryptic peptides of S-[14C]carboxymethyl 14-3-2 yielded only three distinct radioactive peptides, each with one residue of S-carboxymethylcysteine whereas 8 are expected on the basis of the amino acid composition of the 50,000 molecular weight polypeptide chains. Thermolysin digestion of a similarly modified 14-3-2 preparation yielded all of the radioactivity in 5 S-carboxymethylcysteine-containing peptides. The partial amino acid sequence of these peptides indicates that they represent 4 unique areas of the polypeptide chain. Since 8 such peptides were expected, that is, double the number found, the minimum structural unit of the protein must be of 25,000 molecular weight. The results of these experiments do not permit distinction between a duplication of the structure within a single polypeptide chain or the alternate possibility of two polypeptide chains bound by unusually strong non-covalent bonds. These results suggest that 14-3-2 is a covalently linked dimer of 25,000 mol.wt. units that can aggregate to form larger species of 100,000 mol. wt. and higher

Changing the concentration of TEMED in SDS polyacrylamide gels was found to affect the migration of proteins. Elevation of TEMED levels caused a generalized decrease in mobility with some proteins being affected more than others. With various brain protein preparations this differential effect could be used to improve the separation of adjacent protein bands. In additon, it was found that a change in the TEMED concentration affected the results of molecular weight determinations. The effect of TEMED was also observed in one non-SDS system.

Commercially available Coomassie Brilliant Blue R-250 (C.I. 42660) is a popular and useful dye that stains most proteins blue on polyacrylamide gels. Some proteins from brain (rubrophilin), collagens, histones and parotid gland proteins are distinctly red when stained with Coomassie Blue. Commonly used Coomassie Brilliant Blue R-250 preparations may contain more than 30 distinct colored and fluorescent components that can be separated on silica gel chromatographic columns. A specific component has been isolated on silica gel columns that stains rubrophilin and other proline-rich proteins a reddish color. Fast atom bombardment mass spectrometry of the isolated rubrophilin staining principle indicates a molecular weight of 634 as compared to 826 for the major dye in the original Coomassie Brilliant Blue R-250. Infrared spectrometry is consistent with a difference between the rubrophilin staining principle and Coomassie Brilliant Blue R-250 of a toluene sulfonic acid residue.

Amethopterin and 5-bromodeoxyuridine (BUdR) were used to induce morphological changes in cloned rat glioma (C6). The expression of S-100 protein, an acidic protein localized in glial cells, and steroid sulfatase, an ubiquitously distributed enzyme found in high concentration in glial cells, were followed during cell growth, from subculture to well into the stationary phase of control and drug-treated cultures. Amethopterin and BUdR differed in their effects on glioma morphology and in the expression of the biochemical parameters. Amethopterin coordinately stimulated both the production of S-100 protein and steroid sulfatase activity when cell division was inhibited during early logarithmic growth phase. BUdR stimulated steroid sulfatase activity but repressed production of S-100 protein. The results are discussed with respect to the mechanism of regulation of the differentiated state of tumor cells.

The tissue distribution of enzymatic activities in intestinal metaplasia stomachs exhibiting chronic gastritis was compared histochemically with that of the small intestine in man.

Rats were killed after intraventricular administration of [(3)H]leucine, and the turnover rates of total soluble proteins and of the brain specific S-100 protein were determined. The half-life of S-100 was estimated to be 16 days. The S-100 protein, thus, has a turnover not unlike that of the average water-soluble protein of brain.

Publisher Summary This chapter describes the method used in laboratory in the search for brain-specific, species-nonspecific proteins. Protein maps are prepared by fractionating the proteins of aqueous extracts of brain and other organs, such as liver. Presumptively nervous-system-specific proteins are purified from brain by a sequence of steps, such as ammonium sulfate fractionation. Antiserum to the pure protein is prepared by immunizing rabbits and the antiserum is tested for monospecificity by agar diffusion against the pure protein and against brain extract. The antiserum is then used in an immunological assay for the protein—complement fixation. Immunoassays, especially complement fixation, are highly specific and sensitive assays for antigens, being able to detect single amino acid substitutions and to measure the antigens down to the picogram range. The immunoassay can also be used to measure the protein in areas of brain, down to a single cell level, during development, degeneration, and under different functional conditions to localize and determine its function. Preparation of nervous system proteins is explained in the chapter.