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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.

The frequency of flexible ureteroscopy has increased with the introduction of improved instrumentation. Ureteroscopes allow increased endoscopic access to the ureter and kidney. However, maintenance and repair of scopes may increase the total procedure expense.In 3 years (8/2011-7/2014), 655 flexible ureteroscopies were performed at a single-specialty, urology, ambulatory surgery center. Procedures were performed by 26 board-certified urologists using four Olympus URF P5 flexible ureteroscopes. The instruments were handled by a single team and sterilized through the STERIS System E1. Repairs were performed by the manufacturer on an as needed basis. Patient records were reviewed to determine the preoperative diagnosis, operative time, location and size of the stone, and use of laser or ureteral sheath. The occurrence, nature of flexible ureteroscope damage, and cost of repairs were evaluated.Of the ureteroscopies performed, 78% was for the treatment of calculi (50.1% in the kidney). Mean stone size was 8.5 ± 0.2 mm, with larger stones (11 mm) located in the kidney. The flexible ureteroscope was advanced over a guidewire (88% of cases); a laser fiber was introduced in 70%, and a ureteral sheath was used in 13.4%. Mean procedure time was 40 minutes. The most common reasons for ureteroscope repair were cloudy lens (16 repairs) and broken optic fibers (9 repairs). There were 31 repairs during the study period (average 21 cases per repair). Flexible ureteroscopes were out of service for an average of 11 days per repair (range 3-20). The total cost of repairs was $233,150 or ∼$7521 per repair. The average repair cost per flexible ureteroscopy performed was $355.Expenses associated with instrument repair can significantly impact a procedure's net revenue, thus efforts should be made to minimize instrument breakage. The expense of repairing a flexible ureteroscope per procedure can be significant and needs to be considered when pricing this procedure.

We present a series of robotic-assisted laparoscopic ileovesicostomies with bowel work performed completely intracorporeally. The four patients selected for this procedure were all diagnosed with neurogenic bladder and failed conservative medical therapy. Preoperative patient data included age, body mass index (BMI), and urodynamic (UD) study results. Intra-operative data included estimated blood loss (EBL), operative time, and intra-operative complications. Post-operative data included return to bowel function, post-operative complications, and length of hospital stay (LOS). All bowel work was completed intracorporeally with the exception of stoma maturation. Four robotic ileovesicostomies were performed. Pre-operative urodynamic study results showed either elevated detrusor pressures or limited bladder capacities in addition to the inability to perform self-catheterization. The mean patient age was 40 years and mean BMI was 26 kg/m(2). Average EBL and operative time were 131 ml and 290 min, respectively. No intra-operative complications occurred. Bowel function, as defined as flatus, returned on average 3.8 days after surgery and average LOS, defined as discharge home or discharge to the spinal cord unit, was 7.5 days. Mean follow-up time was 25.8 months. Post-operative urodynamic studies revealed low stomal leak point pressure (10 cmH2O). This study is the first to describe a completely intracorporeally robotic-assisted laparoscopic ileovesicostomy with safe and effective outcomes after more than 2 years of follow-up.

both cases, extensive adhesions were found around the kidney, making anatomical identification and dissection difficult. Intra-operative ultrasound and a doppler probe were used to facilitate identification of anatomical structures. The fourth robotic arm and robotic hook were used to help mobilize the kidney and expose important structures. RESULTS: The results of the two cases are presented in Table 1. Both cases were completed robotically with no intraoperative complications. Post-operative course was uneventful and both the patients were discharged on day three. CONCLUSIONS: Salvage robotic kidney surgery for complications of robotic partial nephrectomy is rare but challenging. Intraoperative ultrasound, intraoperative doppler, the fourth robotic arm, and robotic hook canall beuseful todefineanatomyand facilitatedissection indifficult cases.

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.

INTRODUCTION AND OBJECTIVES: Robotic Retroperitoneal Lymphnode Dissection (RPLND) is an emerging option for those undergoing both primary and post-chemotherapy treatment. While open RPLND has previously been the standard of care, however, as surgeons have become more accomplished utilizing robotic assistance so to has this procedure evolved. The post-chemotherapy patient usually presents a specific challenge due to the dense fibrosis associate with this. In this video we present particularly unique case in a patient with duplicated inferior vena cava. Our objective is to demonstrate an interesting and safe means of RPLND utilizing robotic assistance. METHODS: A robotic approach was utilized to perform a bilateral template dissection of the retroperitoneum sparing the right sympathetic chain and superior hypogastric plexus. RESULTS: Further studies are needed to validate the oncologic efficacy of the approach against the standard open approach. CONCLUSIONS: Robotic RPLND in the post-chemotherapy is a safe and feasible alternative to open RPLND with reduced morbidity.

INTRODUCTION AND OBJECTIVES: The apoptotic and suppressive effects of prostate cancer on immune-competent cells are well known. The aim of this study was to evaluate the genome-wide effects of prostate cancer on dendritic cells (DC), the most effective antigenpresenting cells, in a murine prostate cancer model. METHODS: Dendritic cells were compared to DC incubated with RM-1 murine prostate cancer cells. Affymetrix Mouse 430 v2 Array was used for analysis containing 45000 probes. Quantitative polymerase chain reaction (qPCR) was performed to validate changes in the endothelin axis. RNAs from murine prostate cancer and normal murine prostate were also compared. RESULTS: There was a significant increase in the expression of cell death inducing genes in DC incubated with prostate cancer cells, including multiple caspases. There was also a down regulation of proinflammatory genes including a number of interleukins and interferons. A change in the expression of endothelin receptors was seen as well. Considering the role of the endothelin axis in DC functioning, we used qPCR to further evaluate the condition of the endothelin axis in DC under prostate cancer influence. There was a significant decrease in the expression of ET-1 (4.50 1.33 fold) and ETA receptor (2.67 0.13), which are involved in DC survival and pro-inflammatory function. There was also an increase in the expression of ETB receptor (3.42 0.14) involved in DC apoptosis. Genome-wide comparison of RNAs derived from murine prostate cancer and murine prostate further demonstrated decreased expression of apoptotic genes by prostate cancer cells. CONCLUSIONS: Prostate cancer induces a wide variety of changes in DC including decreased expression of genes responsible for pro-inflammatory function and survival. Alterations of the endothelin axis may be one reason why tumor escapes immune monitoring.

The objective of this study is to report the initial results of a newly established robotic prostatectomy (DVP) program at a Veteran's Affairs (VA) medical center. All patients who underwent a radical prostatectomy during the first 18 months of our robotic surgical program were included in this study. These patients were compared to a control group that included all patients who underwent an open prostatectomy 18 months prior to starting our robotic program. Preoperative, intraoperative and postoperative data was compared between open and robotic prostatectomies. 38 men underwent radical retropubic prostatectomy (RRP) between September 2007 and February 2009. With the introduction of robotic prostatectomy, the total number of prostatectomies increased by 84 % to 70 (9 RRP, 61 DVP). Prostate-specific antigen (PSA), Gleason score and clinical stage were similar for both groups. Average estimated blood loss (EBL) was 1205 mL for RRP and 126 mL for DVP. Mean operative times in minutes were 259 and 254 for RRP and DVP, respectively. Complications included two rectal perforations, a cerebrovascular accident, one death after RRP and one open conversion for failure to progress. Average length of stay was 5.1 for RRP and 1.8 days for DVP. Total positive margins were 24 % for RRP and 15 % for DVP. For T2-specific disease, 16.7 % had positive margins after RRP compared to 4.3 % after DVP. The establishment of a robotic prostatectomy program at a tertiary VA medical center was achieved in a safe and efficient manner with improvement in EBL and length of stay when compared to our open prostatectomies. Oncologic outcomes were equivalent when compared to other initial DVP programs.

Introduction The challenge of resident education in urologic surgery programs is to overcome disparity imparted by diverse patient populations, limited training times, and inequalities in the availability of expert surgical educators. Specifically, in the area of prosthetic urology, only a small proportion of programs have full-time faculty available to train residents in this discipline. Aim To examine whether a new model using yearly training sessions from a recognized expert can establish a successful penile prosthetics program and result in better outcomes, higher case volumes, and willingness to perform more complex surgeries. Methods A recognized expert conducted one to two operative training sessions yearly to teach standardized technique for penile prosthetics to residents. Each session consisted of three to four operative cases performed under the direct supervision of the expert. Retrospective data were collected from all penile prosthetic operations before (February, 2000 to June, 2004: N = 44) and after (July, 2004 to October, 2007: N = 79) implementation of these sessions. Main Outcome Measures Outcomes reviewed included patient age, race, medical comorbidities, operative time, estimated blood loss, type of prosthesis, operative approach, drain usage, length of stay, and complications including revision/explantation rates. Statistical analysis was performed using Student's t-tests, Fisher's tests, and survival curves using the Kaplan-Meier technique (P value ≤ 0.05 to define statistical significance). Results Patient characteristics were not significantly different pre- vs. post-training. Operative time and estimated blood loss significantly decreased. Inflatable implants increased from 19/44 (43.2%, pre-training) to 69/79 (87.3%, post-training) (P Conclusions These data demonstrate that yearly sessions with a recognized expert can improve surgical outcomes, type, and volume of implants and can reduce explantation/revision rates. This represents an excellent model for improved training of urologic residents in penile prosthetics surgery.

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.

We used polyclonal antisera recognizing S100, a small acidic protein highly enriched in nervous tissue, to stain sections of embryonic chicken lumbosacral spinal cord and hindlimb. S100 immunoreactivity was detected in developing sensory neurons of the dorsal root ganglia (DRG) and motor neurons of the ventral spinal cord as early as embryonic day (E) 5, and staining persisted through hatching. In contrast, expression of S100 first became apparent in Schwann cells at E13, just before myelination, and was not detected in developing skin or muscle. Since S100 beta was present in motor and sensory neurons and is known to promote neuronal survival and neurite extension in vitro (Winningham-Major, Staecker, Barger, Coats, and Van Eldik, 1989), we tested the ability of S100 to promote neuron survival in an in ovo survival assay. Addition of S100 to chick embryos in ovo during the period of naturally occurring motor neuron cell death resulted in a significant increase in motor neuron survival, but had no effect on the in vivo survival of sensory neurons in the DRG. The findings that S100 is present in spinal motor neurons and that the addition of S100 enhances the survival of these cells in vivo are consistent with the possibility that S100 may act as a naturally occurring neuron survival factor during development.

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.

Since 2003 the increasing use of robotic-assisted laparoscopic prostatectomy has been accompanied by the need to be prepared for a new set of problems in the operating room. Operative complications unique to the robot and its components are rare but can lead to case conversion and procedural abandonment. We describe an innovative solution to the uncommon problem of intraoperative robotic light source failure. Surgeons carrying out such procedures should be aware of this complication and be able to substitute a comparable light source. Possession of an appropriate type of low-cost alternative light source could prevent unnecessary procedural abandonment or open conversion in the setting of mid-operative light source failure.

— Several major brain-specific proteins have been detected in cephalopods by electrophoretic analysis of the soluble proteins extracted from the optic lobes and other organs of octopus and by 2-dimensional fractionation of the soluble proteins from optic lobes and hepatopancreases of octopus and squid. One of the brain-specific proteins from octopus, identified as 0-1, has been purified by chromatography on DEAE-cellulose, Sephadex G-150, and DEAE-Sephadex. The protein appears to be pure on the basis of several physicochemical criteria. Amino acid analysis indicates a high content of glutamic and aspartic acids or their amides (or both) and the lack of tryptophan. A molecular weight of 17,000 has been calculated from sodium dodecyl sulphate-gel electrophoresis, gel filtration and ultracentrifugation analysis. The preparation of a specific rabbit antiserum against 0-1 has allowed its determination by agar immunodiffusion and complement fixation techniques. With the latter procedure it has been shown that the protein is absent outside the nervous system, is present in a concentration of several mg/g wet weight in octopus brain and is widely distributed within the octopus central and peripheral nervous system and in several molluscan species. It is also present in optic lobes of octopus at early stages of development.

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.

The distribution of S-100 outside the central nervous system in humans and rats was explored using antiserum to S-100 and the peroxidase anti-peroxidase method of Sternberger. In peripheral nerves the Schwann cells and the outermost part of the myelin sheaths were stained; axons were not. In dorsal root ganglia and ganglia of the autonomic nervous system only satellite cells were stained. In the adrenal medulla a considerable number of cells were stained. In all other organs studied Schwann cells and satellite cells of ganglia were the only elements that were stained. We conclude that S-100 could serve as a marker for Schwann cells in situ.

A brain specific protein, 14-3-2, has been isolated from bovine brain by the use of ammonium sulfate precipitation, gel filtration on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose and DEAE-Sephadex. It is an acidic protein, in agreement with the high content of glutamic and aspartic acid, that is composed of a single polypeptide chain of mol. wt. 50,000. Immunochemical tests using antiserum to purified 14-3-2 showed that the protein is present in at least 100-fold greater amounts in brain than in any other rat tissue. Furthermore, 14-3-2 was found in brains of a number of vertebrate species, although the antigen is apparently not entirely identical in all species tested. The protein 14-3-2 can be considered to be a species non-specific protein which is neuronal in origin.

Water-soluble proteins were extracted from individual retinas, optic nerves, combined optic tracts and lateral geniculate bodies, and superior colliculi of rabbits at 1, 3, and 18 days after injection of [3H]leucine into the right eye. The Ca2+-dependent protein modulator of 3′:5′-cyclic-AMP phos-phodiesterase (calmodulin) was isolated from these samples by a two-step polyacrylamide gel electrophoresis procedure. An analysis of the radioactivity incorporated into the total soluble proteins and the calmodulin revealed that most of the calmodulin was axonally transported at a slow rate (2–4 mm/day) and represented about 0.45% of the total transported soluble protein.

Rats undernourished from birth to 28 days of age demonstrated decreases in the concentration of the glial specific protein S-100. The concentration of S-100 in cerebral cortex was 77% and 74% that of well-fed controls at 21 and 28 days of age, respectively, while in the diencephalon the concentration of S-100 was 68% that of the controls at 28 days of age. In contrast, the concentration of 14-3-2, a neuronal protein, does not differ from controls in any area of brain at 21 days of age.

The protein organization of rat brain synaptic plasma membranes (SPM) and synaptic vesicles (SV) was investigated by surface iodination and one- and two-dimensional electrophoresis. Polypeptides of molecular weights (MWs, in Kilodaltons) 170 K, 135 K, 96-86 K, 68-64-61 K, 56 K, 52 K, 38 K, 35-33 K, and 18 K are predominantly or exclusively exposed on the extracellular side of synaptosomes. Several polypeptides of MW between 70 K and 40 K are exclusively exposed on the cytoplasmic side of SPM. The use of two-dimensional electrophoresis allowed to recognize that, for some classes of MW, there are polypeptides of nearly the same MW and different isoelectric points exposed on both sides of SPM. The synaptosomal membrane shows a predominance of acidic proteins on the extracellular side and more neutral and basic proteins on the cytoplasmic side. With respect to SPM, SV are particularly enriched with polypeptides of MW 71 K, 56 K, 39-38 K, 32 K, 16 K, and 15 K. One of them, a doublet of MW 39-38 K, is the most highly labeled species upon surface iodination and is similar, but not identical, with a doublet located on the cytoplasmic side of SPM.

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.

Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into superfusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S-100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 micrograms S-100/mg protein, respectively. These values were three- to sixfold higher than the S-100 content of the soluble cytoplasmic protein fractions from the same tissue. This several-fold higher S-100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S-100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.

An assay, based on complement fixation, was described for the S-100 protein, a protein characteristic of the nervous system. It was found to be distributed in all parts of the nervous system both peripherally and centrally. It was not possible to deduce from the distribution the localization in cell type, but in human brain the concentration in each of twenty-six areas was consistent from brain to brain.

An attempt has been made to localize, at a cellular level, the two brain specific proteins, S-100 and 14-3-2, by determining their relative concentrations in thalamic nuclei undergoing retrograde cell degeneration after appropriate lesions of the cerebral cortex. As the neuronal degeneration progresses the concentration of the 14-3-2 protein declines so that 6 weeks postoperatively it is only approximately 40% of its control level. On the other hand, there is a slight, but statistically significant, increase in the level of the S-100 protein between the 1st and 4th postoperative weeks which appears to be correlated with the early gliosis in the affected nuclei. On this basis it is suggested that 14-3-2 is primarily a neuronal protein and that the S-100 protein is largely, if not exclusively, confined to glial cells.

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.

Summary The concentrations of the two brain specific proteins, 14-3-2 which is predominantly neuronal, and S-100 which is glial, have been determined in the optic tectum of a series of chick embryos between the 3rd day of incubation and the 9th week post-hatching. Both proteins first appear during the 4th day of incubation at about the time the first differentiated cells can be recognized morphologically. The level of the two proteins shows little increase during the ensuing 8 days at which time cell proliferation ceases, but between about the 17th day of incubation and the 2nd week after hatching the concentration of both proteins increases more-or-less exponentially. Thereafter the rate of increase declines rapidly, but there is a slow continuing increase for some weeks until adult levels are reached. Although the changes in concentration of the two proteins show a close parallelism, the concentration of 14-3-2 is approximately 100 times greater than that of the S-100 protein at each stage in development.

1. 1. Hemolysis of rat erythrocytes by all concentrations employed of phenylmercuric compounds begins immediately without a lag period. 2. 2. The hemolysis is inhibited by glucose both by delaying its onset and by slowing its progress after it has begun. 3. 3. Certain other sugars also have a similar effect. 4. 4. Possible mechanisms are discussed.

Summary A micro complement-fixation assay is described which is sensitive to 1 to 10 × 10-12 g of antigen. The method was shown to be capable of measuring a specific antigen in brain extracts made from amounts of brain (dry weight) as small as 1.4 × 10-9 g, and in extracts from layers of cerebellum.

— —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.

Purified nerve growth factor induced the outgrowth of neurites from cultured human neuroblastoma cells (NJB line) and a concomitant increase in colchicine-binding activity in extracts from these cultures. The parallel stimulation of neurite outgrowth from the cells and colchicine-binding activity of the extracts is interpreted to represent de novo synthesis of microtubular subunit protein in response to the challenge by nerve growth factor. The regulation of the expression of 14-3-2 protein, a protein characteristic of differentiated neuronal cells, was not affected in NJB cells by the addition of nerve growth factor to the culture medium. 14-3-2 protein is present in NJB cells at a concentration equal to that present in human brain from subculture to the stationary phase of growth of the tumor cells. It was concluded that these two gene products, characteristic of differentiated neural cells, are not coordinately regulated in NJB human neuroblastoma cells.

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.

A major glial structure is present during development within the midline raphe of the midbrain, hindbrain, and cervical spinal cord of the rat. It is composed of great numbers of glial cell bodies lying immediately ventral to the cerebral ventricular system and the large radial processes extending from these cells toward the ventral surface of the brain roughly within the midsagittal plane. There is also a smaller group of glial cells on the dorsal surface of the aqueduct and the central canal whose processes extend to the dorsal surface of the brain. The entire structure exhibits an intensely positive immunoreactivity with the antibody to the S-100 protein, a nervous-system-specific protein found primarily in the cytoplasm of astrocytes. This immunoreactivity makes possible a clear visualization of the extent, magnitude, and continuity of this structure from at least embryonic day 15, the first age examined, until postnatal days 7–8, when it is no longer visible by this technique. This glial structure has several prominent morphological characteristics. During prenatal and early postnatal development the fibers forming the ventral aspect of the structure in the midbrain and hindbrain are formed into two parallel plates on either side of the midline with S-100-negative tissue between the plates. As development progresses, S-100-positive fibers are continually added so that the plates become thicker at the expense of the nonstaining intervening area. By postnatal day 4 only a single midline plate of fibers is visible, occupying the entire midline raphe. In the region of the pontine flexure the entire structure takes on a distinctly pleated configuration. This fact produces a curious “sine wave” appearance when the plane of section crosses these vertical pleats. At postnatal day 5 the structure begins to disappear, and it is no longer visible by 7–8 days postnatal. This glial structure does not stain with antisera to glial fibrillary acidic protein, a protein associated with fibrous astrocytes, or routine cell stains such as cresyl violet. With these techniques the raphe area appears essentially devoid of identifiable cellular elements.

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.