Dye Column Research Articles

Potential of nanocomposites of zero valent copper and magnetite with Eleocharis dulcis biochar for packed column and batch scale removal of Congo red dye

The current study is the first attempt to prepare nanocomposites of Eleocharis dulcis biochar (EDB) with nano zero-valent Copper (nZVCu/EDB) and magnetite nanoparticles (MNPs/EDB) for batch and column scale sequestration of Congo Red dye (CR) from synthetic and natural water. The adsorbents were characterized with advanced analytical techniques. The impact of EDB, MNPs/EDB and nZVCu/EDB dosage (1–4 g/L), pH (4–10), initial concentration of CR (20–500 mg/L), interaction time (180 min) and material type to remove CR from water was examined at ambient temperature. The CR removal followed sequence of nZVCu/EDB > MNPs/EDB > EDB (84.9–98% > 77–95% > 69.5–93%) at dosage 2 g/L when CR concentration was increased from 20 to 500 mg/L. The MNPs/EDB and nZVCu/EDB showed 10.9% and 20.1% higher CR removal than EDB. The adsorption capacity of nZVCu/EDB, MNPs/EDB and EDB was 212, 193 and 174 mg/g, respectively. Freundlich model proved more suitable for sorption experiments while pseudo 2nd order kinetic model well explained the adsorption kinetics. Fixed bed column scale results revealed excellent retention of CR (99%) even at 500 mg/L till 2 h when packed column was filled with 3.0 g nZVCu/EDB, MNPs/EDB and EDB. These results revealed that nanocomposites with biochar can be applied efficiently for the decontamination of CR contaminated water.

Adsorptive column studies for removal of Reactive Blue 4 dye using dry cells of Rhizopus oryzae (MTCC 262)

The biosorption potential of dry biomass of Rhizopus oryzae (MTCC 262) in removing a textile dye Reactive Blue 4 from dye contaminated wastewater using up-flow packed-bed column reactor has been studied. The impact of operating parameters like flow rate of dye solution, bed height of column and concentration of dye in the feed solution on the removal potential has also been studied. The Adam-Bohart Model, Yoon-Nelson Model and Thomas model are used for the analysis of experimental data. Dye removal of 71.81% is obtained using dry biomass at flow rate of 2 mL min-1of dye solution, bed height of 15 cm and influent dye concentration of 200 mg L-1. Adsorbed dye can be successfully recovered from dry R.oryzae (MTCC 262) biomass of packed bed column reactor by running 1 (N) NaOH solution. Results demonstratesignificant reduction in Reactive Blue 4 concentration after the biosorption process.

Lumbar erector spinae plane block: an anatomical and dye distribution evaluation of two ultrasound-guided approaches in canine cadavers

ObjectiveTo examine the anatomy of the lumbar epaxial region and to describe two different ultrasound-guided approaches for the lumbar erector spinae plane (ESP) block in dogs. Study designAn anatomical and experimental cadaver study. AnimalsA group of 19 canine cadavers. MethodsThe anatomy was described following dissection of two cadavers. Bilateral ultrasound-guided ESP injections with 0.4 mL kg–1 of contrast dye were performed in 17 adult Beagle cadavers using either transversal (TVS) or parasagittal (PST) approaches. Computed tomography was performed to measure the total length of the contrast dye column and the epidural, intravascular, hypaxial and intra-abdominal migration. Dissections were performed to assess the spread of the contrast dye and to determine the degree of staining of the dorsal branches of the spinal nerves (DBSN). Mann–Whitney U and chi-square tests were used to compare data between groups. ResultsUsing both techniques, the contrast dye was observed within the ESP compartment. There was no difference in the total length of the contrast dye column between TVS and PST approaches (p = 0.056). Using the TVS approach, multisegmental staining of the DBSN was visible with 100% (17/17) of injections, while complete staining of the DBSN was achieved at 94% of the injection sites. Using the PST approach, these values were 29% (5/17) and 23% (4/17), respectively. The TVS approach stained more DBSN than the PST approach (p = 0.001), with a median (range) of 2 (2–3) versus 0 (0–3) DBSN, respectively. Using the TVS approach, epidural and intravascular migration were present in 2/17 (p = 0.485) and 3/17 (p = 0.227) injections, respectively. Conclusions and clinical relevanceBoth ultrasound-guided approaches resulted in a spread of the contrast dye within the ESP compartment. Although there were no differences in the total length of the contrast dye column, the TVS approach was superior to the PST approach in staining DBSN.

Abstract 2851: A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format

Abstract A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format. Removal of unreacted or residual compounds in the sample-preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non-specific binding of free-dye resulting in high background issues during fluorescent imaging. Commonly used clean-up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size-exclusion chromatography requires expensive instrumentation in addition to lengthy set up time. We have developed the Thermo Scientific™ PierceTM Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL- 4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non-conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above-mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered. Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering >90% of the added protein. Results also indicate >90% removal of biotinylated molecules and crosslinkers from Protein samples with >90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO-1 monoclonal antibody conjugated Alexa Fluor™ 488 which was purified using PierceTM Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco-2 cells than the non-purified conjugate. Citation Format: Deven Kent Etnyre, Ramesh Ganapathy, Marie Nlend, Chris Etienne, Atul M. Deshpande. A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2851.

Comparison of the clinical and radiographic appearance of the cervical vertebrae with histological and anatomical findings in an eight-month old warmblood stallion suffering from cervical vertebral stenotic myelopathy (CVSM)

BackgroundCervical vertebral stenotic myelopathy (CVSM) remains one of the most important abnormalities of the cervical spine resulting in neurological deficits in horses. The aim of the following study was to compare the results of the clinical and neurological examination, the results of myelography and the post mortem anatomical and histological appearance of the spinal cord and cervical vertebrae in a horse with CVSM.Case presentationThe following study describes a clinical case of an eight-month-old stallion with ataxia. Plain cervical radiographs indicated narrowing of the spinal canal. Conservative therapy using NSAIDs did not result in any improvement in the gait of the horse. Due to economic constraints, surgical intervention was excluded. The owner chose to humanely euthanise the horse. Immediately after euthanasia, post mortem myelography was performed, and measurements of the myelographic dye column were taken. They revealed a 67% DMC reduction and a 64% DD reduction at the C3/C4 level. Afterwards, an anatomical dissection was performed. The cervical vertebrae and vertebral canal were macroscopically inspected and measured and indicated a 44% narrowing of the canal at the C3/C4 level. The spinal cord was removed and underwent histological evaluation after staining. Microscopic lesions were visible at the level of the compression and included axonal degeneration with partial or complete loss of myelin in the white matter of the lateral and dorsal funiculi as well as the formation of dysfunctional so-called “spongy structures”. An increase in the number of microglial cells and collagen was also observed. The formation of glial scars was excluded. Immunohistochemical studies revealed a negative transmembrane glycoprotein CD68(−) – monocyte response and a negative tumor necrosis alpha TNFα (−) reaction.ConclusionsCVSM may be difficult to diagnose, even for experienced veterinary surgeons. Currently, an ex vivo histopathologic examination of the spinal cord is thought to be the gold standard in the diagnosis of CVSM. Our histological examination revealed no CVSM-specific glial scar formation and a CD68(−) negative and TNF-α negative reaction, which have not been previously reported. Histological lesions in CVSM may vary depending show inter-individual variability and on the treatment, which further hinders ex-vivo diagnostics.

Column, kinetic and isotherm studies of PAH (phenanthrene) and dye (acid red) on kaolin modified with 1-hexyl, 3-decahexyl imidazolium ionic liquid

Adsorption studies were carried out to determine the efficiency of kaolin modified with a novel long chain ionic liquid (IL) 1-hexyl, 3-decahexyl imidazolium. Synthesis of IL was carried out and characterised with NMR and fourier transform infrared (FT-IR) spectroscopy. Kaolin was modified with IL to form hydrophobic kaolin (modified–kaolin) by adding IL in excess of the cation exchange capacity of kaolin. Modified–kaolin was characterised using FT-IR, x-ray diffraction (XRD), Brunauer−Emmett−Teller (BET), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM). Modified–kaolin was used in column and batch adsorption studies for phenanthrene and acid red dye. Modified–kaolin was an efficient material for the removal of phenanthrene and acid red dye. For column studies, the adsorption capacity, depth of exchange zone, time required for the pollutant to move through the exchange zone which is the height of the adsorbent in the column and adsorption rates were investigated using the Adams-Bohart, Yoon-Nelson model flow and Thomas sorption model. Column adsorption capacity at the same flow rate (5.0mLmin−1) and different concentrations (50, 100 and 200mgL−1) for phenanthrene were 222.9, 611.8 and 1093.5mgg−1, while that of acid red dye were 877.0, 1337.4 and 1350.7mgg−1, respectively. Thomas and Yoon–Nelson models best explained the column studies. Data from both batch studies fitted well to pseudo-second order. The Langmuir isotherm had the best fit with an adsorption capacity of 842.7mgg−1 for dye and the Freundlich isotherm best described phenanthrene with an adsorption capacity of 188.9mgg−1.

Shear Strength of Connections between Concrete Filled Steel Tubes and Beams

Departure from the stress state of the steel pipe concrete dye column node, axial force, steel pipe column end moments and stained end vertical shear strength Shear strength concrete filled steel tube the formula, using the finite element analysis software ANSYS to establish a three-dimensional nonlinear finite element model, comparing the results calculated by the finite element calculations and formulas seen, the model is applied to the concrete filled steel tube - steel beam finite element nodes analysis.

Apnea and unconsciousness after accidental subdural placement of an epidural catheter

Apnea and unconsciousness after accidental subdural placement of an epidural catheter

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-myristoylglucosamine and acetate in Gram-negative bacteria. This second, and committed, step in lipid A biosynthesis is a target for antibiotic development. LpxC was previously identified as a mononuclear Zn(II) metalloenzyme; however, LpxC is 6-8-fold more active with the oxygen-sensitive Fe(II) cofactor (Hernick, M., Gattis, S. G., Penner-Hahn, J. E., and Fierke, C. A. (2010) Biochemistry 49, 2246-2255). To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with K(D) values of 60 ± 20 pM and 110 ± 40 nM, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.

Diagnosis and surgical treatment of epidural neoplasms in two ataxic horses

SummaryTwo cases of ataxia caused by epidural neoplasia diagnosed with myelography and histopathology are presented in this report. One horse was diagnosed with lymphohistiocytic lymphosarcoma and the other with haemangiosarcoma. In both cases, subtotal dorsal decompression was performed as part of their treatment protocol. One horse was subjected to euthanasia immediately after surgery; however, the other horse recovered well, with no gait abnormalities and a normal myelographic dye column seen at 6 months post surgery. Although both horses were ultimately subjected to euthanasia, subtotal dorsal decompression provides a potential surgical treatment for epidural neoplasia.

A model‐dye comparison experiment in the tidal mixing front zone on the southern flank of Georges Bank

A process‐oriented model‐dye comparison experiment was conducted to examine the ability of a numerical ocean model to simulate the observed movement of dye across the tidal mixing front on the southern flank of Georges Bank during 22–26 May 1999. The experiment was made using the unstructured‐grid Finite‐Volume Coastal Ocean Model (FVCOM) with varying horizontal resolution. The results indicate that the observed cross‐isobath movement of the dye patch was primarily controlled by meso‐scale temporal and spatial variability of the water temperature and salinity fields. Onset of vertical stratification tended to slow down an upward stretching of the dye column and trapped the dye within the bottom mixed layer. To reach a convergent numerical solution that reproduced the observed lateral turbulent dispersion of dye, the FVCOM grid required a horizontal resolution of ∼500 m in the dye study region. Within the tidal mixing front of Georges Bank, the movement of the center of the dye patch was mainly driven by the ensemble velocity integrated over the dye volume, with a first‐order contribution from vertical shear of the dye's horizontal velocity.

Intraoperative Technique to Define the Safe Lateral Limits of Anterior Cervical Corpectomy

We describe a surgical radiographic technique during anterior cervical corpectomy to define the safe lateral limit of dissection. To assess the radiographic technique accuracy using computed tomography (CT) measurements on cadaveric specimens. Two clinical cases are presented. During anterior cervical surgery loss of orientation can lead to eccentric decompression and vertebral artery injury. A two-level corpectomy on 4 cadaveric samples was filled with radioopaque dye. An "introperative" antero-posterior x-ray was used to measure the narrowest distance from dye column to uncovertebral joints. A CT scan confirmed the distance from corpectomy to vertebral arteries. The distance between the x-ray dye column and uncovertebral joints averaged 2.7 mm (range, 0 to 7+/-2.2 mm). A CT scan demonstrated the distance from corpectomy to vertebral artery averaged 4.5 mm (range, 0 to 10+/-4.4 mm). The measured distance underestimated the true distance by an average of 1.8 mm (range, 0 to 8+/-2.2 mm). An intraoperative radiographic technique can estimate the lateral distance between the corpectomy and vertebral arteries by measuring the distance from the dye column to uncovertebral joints. The dye radiographic technique provides the surgeon an additional margin of safety.

Process development for the removal and recovery of hazardous dye erythrosine from wastewater by waste materials—Bottom Ash and De-Oiled Soya as adsorbents

Erythrosine is a water-soluble xanthene class of dye. It is widely used as colorant in foods, textiles, drugs and cosmetics. It is highly toxic, causes various types of allergies, thyroid activities, carcinogenicity, DNA damage behaviour, neurotoxicity and xenoestrogen nature in the humans and animals. The photochemical and biochemical degradation of the erythrosine is not recommended due to formation of toxic by-products. The present paper is an attempt to remove erythrosine from wastewater using adsorption over Bottom Ash—a power plant waste and De-Oiled Soya—an agricultural waste. Under the batch studies, effect of concentration of dye, temperature, pH of the solution, dosage of adsorbents, sieve size of adsorbents, etc., have been studied for the uptake of the dye over both adsorbents. The adsorption process verifies Langmuir and Freundlich adsorption isotherms in both the cases and based on the data different thermodynamic parameters have been evaluated. Batch studies also include kinetic measurements, rate constant study, mass transfer behaviour and establishment of mechanistic pathway for both the cases. For the bulk removal of the dye column operations have been carried out and breakthrough capacities of the Bottom Ash and De-Oiled Soya columns have been calculated. Attempts have also been made for the recovery of the adsorbed dye from exhausted columns by eluting dilute NaOH and more than 90% of the dye was recovered.

Epidural migration of new methylene blue in 0.9% sodium chloride solution or 2% mepivacaine solution following injection into the first intercoccygeal space in foal cadavers and anesthetized foals undergoing laparoscopy

To determine the relationship between epidural cranial migration and injectate volume of an isotonic solution containing dye in laterally recumbent foal cadavers and evaluate the cranial migration and dermatome analgesia of an epidural dye solution during conditions of laparoscopy in foals. 19 foal cadavers and 8 pony foals. Foal cadavers received an epidural injection of dye solution (0.05, 0.1, 0.15, or 0.2 mL/kg) containing 1.2 mg of new methylene blue (NMB)/mL of saline (0.9% NaCl) solution. Length of the dye column and number of intervertebral spaces cranial and caudal to the injection site were measured. Anesthetized foals received an epidural injection of dye solution (0.2 mL/kg) containing saline solution or 2% mepivacaine. Foals were placed in a 100 head-down position, and pneumoperitoneum was induced. Dermatome analgesia was determined by use of a described electrical stimulus technique. Foals were euthanatized, and length of the dye column was measured. Epidural cranial migration of dye solution in foal cadavers increased with increasing volume injected. No significant difference was found in epidural cranial migration of a dye solution (0.2 mL/kg) between anesthetized foals undergoing conditions of laparoscopy and foal cadavers in lateral recumbency. Further craniad migration of the dye column occurred than indicated by dermatome analgesia. Epidural cranial migration increases with volume of injectate. On the basis of dermatome analgesia, an epidural injection of 2% mepivacaine (0.2 mL/kg) alone provides analgesia up to at least the caudal thoracic dermatome and could permit caudal laparoscopic surgical procedures in foals.

Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns.

The reliable identification and analysis of the low abundance proteins expressed by a cell remains a key challenge in the study of cellular proteomes. The analysis of low abundance proteins is a particular problem when using two-dimensional gel electrophoresis (2-DE) to resolve the cellular proteins since the technology is unable to display the wide dynamic range of protein levels typically synthesized by cells. We have investigated the use of reactive dye compounds for the enrichment of low abundance cellular proteins prior to analysis by 2-DE. The capacity of reactive dye compounds to bind specific protein species was used as the basis for a general chromatographic tool for protein enrichment. Six reactive dye compounds were investigated in detail for the analysis of Escherichia coli proteins. Whole bacterial cell lysates were passed down columns prepared with the reactive dye compounds. The bound proteins were eluted with 1.5 M NaCl and analyzed by 2-DE. Distinctive protein profiles were observed for the bound proteins recovered from the different reactive dye compounds. Selected proteins enriched by these methods were identified by peptide mass mapping. The enrichment procedure developed using reactive dye compounds were used to investigate acid-induced changes in the proteome of E. coli grown at either pH 7.0 or pH 5.8. Increased levels of expression were observed for a number of proteins (for example, GdhA, PanC, ProC, TkrA, EF-TS and YodA) were observed for E. coli grown at pH 5.8. Five identified proteins (AroG, FabI, GlyA, PurA and EF-Tu) showed reduced levels of synthesis for bacteria grown at pH 5.8 compared to pH 7.0. In the case of PanC and FabI the altered expression profiles were only reliably demonstrated using the enrichment protocols. One theme emerging from these data was that the expression of proteins concerned with one-carbon metabolism was perturbed at pH 5.8, which may point to a previously unrecognized affect of low pH stress on the physiology of E. coli cells. We conclude that the prefractionation of cell lysates on reactive dye columns will serve as a valuable generic tool for the analysis of low abundance proteins expressed by both prokaryotic and eukaryotic cells.

Purification and inactivation of 3-hydroxyanthranilic acid 3,4-dioxygenase from beef liver

3-Hydroxyanthranilic acid 3,4-dioxygenase (EC 1.13.11.6; HADO) was purified to homogeneity from beef liver with the use of two dye columns (Cibacron Blue and Reactive Green 19) and hydroxyapatite. Two active peaks of enzyme were isolated from the hydroxyapatite column or by nondenaturing chromatofocusing of the enzyme prior to hydroxyapatite. The two active forms moved with different electrophoretic mobilities when they were subjected to nondenaturing polyacrylamide gel electrophoresis, regardless of the method of isolation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), however, these species had apparently identical mobilities and have, therefore, close molecular mass. Analysis by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry gave them a molecular mass of 32,566 and 32,515 Da, respectively, for the species with apparent p I values of 5.60 and 4.98, respectively, suggesting that they differ only in the presence or absence of the iron cofactor. The N-terminal group appears to be blocked as no amino-terminal sequence was possible from direct Edman degradation. A new inactivator of the enzyme, 6-chloro-3-hydroxyanthranilic acid, was synthesized and was shown to exhibit time-dependent inactivation. A possible mechanism for inactivation is proposed.

Purification of Muscle Enzymes by Pseudoaffinity Chromatography

ABSTRACT A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.

Definition of the prostate in CT and MRI: a multi-observer study

Purpose: To determine, in three-dimensions, the difference between prostate delineation in magnetic resonance (MR) and computer tomography (CT) images for radiotherapy treatment planning. Patients and Methods: Three radiation oncologists, considered experts in the field, outlined the prostate without seminal vesicles both on CT, and axial, coronal, and sagittal MR images for 18 patients. To compare the resulting delineated prostates, the CT and MR scans were matched in three-dimensions using chamfer matching on bony structures. The volumes were measured and the interscan and interobserver variation was determined. The spatial difference between delineation in CT and MR (interscan variation) as well as the interobserver variation were quantified and mapped three-dimensionally (3D) using polar coordinates. A urethrogram was performed and the location of the tip of the dye column was compared with the apex delineated in CT and MR images. Results: Interscan variation: CT volumes were larger than the axial MR volumes in 52 of 54 delineations. The average ratio between the CT and MR volumes was 1.4 (standard error of mean, SE: 0.04) which was significantly different from 1 ( p < 0.005). Only small differences were observed between the volumes outlined in the various MR scans, although the coronal MR volumes were smallest. The CT derived prostate was 8 mm (standard deviation, SD: 6 mm) larger at the base of the seminal vesicles and 6 mm (SD 4 mm) larger at the apex of the prostate than the axial MRI. Similar figures were obtained for the CT and the other MRI scans. Interobserver variation: The average ratio between the volume derived by one observer for a particular scan and patient and the average volume was 0.95, 0.97, and 1.08 (SE 0.01) for the three observers, respectively. The 3D pattern of the overall observer variation (1 SD) for CT and axial MRI was similar and equal to 3.5 to 2.8 mm at the base of the seminal vesicles and 3 mm at the apex. Conclusion: CT-derived prostate volumes are larger than MR derived volumes, especially toward the seminal vesicles and the apex of the prostate. This interscan variation was found to be larger than the interobserver variation. Using MRI for delineation of the prostate reduces the amount of irradiated rectal wall, and could reduce rectal and urological complications.

Localization of the prostatic apex for radiotherapy treatment planning using urethroscopy

To assess the ability of computed tomography (CT) scans and retrograde urethrograms to accurately define the prostatic apex in the craniocaudad dimension, using urethroscopy as a reference. Plain film radiographs of the pelvis were obtained in 15 patients with early-stage adenocarcinoma of the prostate, with the tip of a urethroscope held in place at the external sphincter, which most closely approximates the prostatic apex. The scope was then withdrawn, and a retrograde urethrogram was performed. Immediately afterwards, a CT scan of the pelvis was obtained. Because differential filling of the bladder and rectum affects the position of the prostatic apex, patients voided prior to rather than in between the three consecutive studies. The urethroscopy-defined prostatic apex was located 4 +/- 8 mm (mean +/- SD) superior to the CT-defined apex, 13 +/- 3 mm (mean +/- SD) superior to the urethrogram tip and 30 +/- 7 mm (mean +/- SD) superior to the ischial tuberosities. There was significant interobserver variability in the location of the prostatic apex as determined by CT scans. Placement of the inferior border of the radiation portals at the ischial tuberosities would have resulted in irradiation of > or = 20 mm bulbar urethra, as defined by the dye column of the retrograde urethrogram, in 6 out of 15 (40%) of the patients and irradiation of < 10 mm bulbar urethra in 2 out of 15 (13%) of the patients. Because the prostate blends inferiorly with the urogenital diaphragm, CT scans do not allow one to precisely localize the prostatic apex. Due to anatomic variability, the ischial tuberosities do not allow one to accurately localize the prostatic apex. Retrograde urethrograms provide helpful supplemental information regarding the position of the prostatic apex for radiotherapy treatment planning.

Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M r on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M r of 87 000 ± 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD + completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients s 20,w of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M r,app of 280 000 ± 5000 g/mol, consistent with the hexameric structure for the wild-type protein and 135 000 ± 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dinner (98 000) and a trimer (147 000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M r,app showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 1/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 1/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.