Assessment of hydroxylated metabolites of polychlorinated biphenyls as potential xenoestrogens: a QSAR comparative analysis∗.

Alternative methods, including quantitative structure-activity relationships (QSAR), are being used increasingly when appropriate data for toxicity evaluation of chemicals are not available. Approximately 40 mono-hydroxylated polychlorinated biphenyls (OH-PCBs) have been identified in humans. They represent a health and environmental concern because some of them have been shown to have agonist or antagonist interactions with human hormone receptors. This could lead to modulation of steroid hormone receptor pathways and endocrine system disruption. We performed QSAR analyses using available estrogenic activity (human estrogen receptor ER alpha) data for 71 OH-PCBs. The modelling was performed using multiple molecular descriptors including electronic, molecular, constitutional, topological, and geometrical endpoints. Multiple linear regressions and recursive partitioning were used to best fit descriptors. The results show that the position of the hydroxyl substitution, polarizability, and meta adjacent un-substituted carbon pairs at the phenolic ring contribute towards greater estrogenic activity for these chemicals. These comparative QSAR models may be used for predictive toxicity, and identification of health consequences of PCB metabolites that lack empirical data. Such information will help prioritize such molecules for additional testing, guide future basic laboratory research studies, and help the health/risk assessment community understand the complex nature of chemical mixtures.

Effects of supplementation on intake, digestion, and performance of beef cattle consuming fertilized, stockpiled bermudagrass forage.

Experiments were conducted to determine the effects of increasing supplement protein concentration on performance and forage intake of beef cows and forage utilization of steers consuming stockpiled bermudagrass forage. Bermudagrass pastures were fertilized with 56 kg of N/ha in late August. Grazing was initiated during early November and continued through the end of January each year. Treatments for the cow performance trials were: no supplement or daily equivalents of 0.2, 0.4, and 0.6 g of supplemental protein per kilogram of BW. Supplements were formulated to be isocaloric, fed at the equivalent of 0.91 kg/d, and prorated for 4 d/wk feeding. Varying the concentration of soybean hulls and soybean meal in the supplements created incremental increases in protein. During yr 1, supplemented cows lost less weight and condition compared to unsupplemented animals (P < 0.05). During yr 2, supplemented cows gained more weight (P = 0.06) and lost less condition (P < 0.05) compared to unsupplemented cows. Increasing supplement protein concentration had no affect on cumulative cow weight change or cumulative body condition score change. Forage intake tended to increase (P = 0.13, yr 1 and P = 0.07, yr 2) in supplemented cows. Supplement protein concentration did not alter forage intake. In a digestion trial, four crossbred steers were used in a Latin square design to determine the effects of supplement protein concentration on intake and digestibility of hay harvested from stockpiled bermudagrass pasture. Treatments were no supplement; or 0.23, 0.46, and 0.69 g of supplemental protein per kilogram of BW. Forage intake increased (P < 0.05) 16% and OM intake increased (P < 0.01) 30% in supplemented compared to unsupplemented steers. Diet OM digestibility increased (P = 0.08) 14.5% and total digestible OM intake increased (P < 0.05) 49% in supplemented compared to unsupplemented steers. Supplement protein concentration did not alter forage intake, total digestible OM intake, or apparent digestibility of OM or NDF. During the initial 30 d after first killing frost, beef cows did not respond to supplementation. However, later in the winter, supplementation improved utilization of stockpiled bermudagrass forage.

Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme.

A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage.

Modified alpha wrap techniques for dynamic urethral graciloplasty in an animal model.

Urethral sphincter reconstruction with a stimulated skeletal muscle flap has been used for treatment of severe intrinsic sphincter deficiency. Urethral strictures and failures were reported in some of the initial experiences. The etiology of these problems is not known, but elevated resting urethral pressures and excessive urethral displacement with stimulation are possible causes. We modified two operative techniques in forming dynamic urinary graciloplasty (DUG) in an attempt to minimize resting urethral pressure without stimulation and urethral mobility during stimulation. Two types of DUG were used. In the first group, a small flap (partial muscle wrap) from the gracilis muscle with an attachment site on the muscle was constructed in four dogs. In the second group, three dogs with a modified alpha wrap and proximal attachments were used. All of the gracilis muscle wraps were stimulated using an implanted programmable pulse stimulator with electrodes attached over the motor nerve. Following a 2-week, postrecovery period, urethral pressure measurements were obtained with and without stimulation. Five weeks were used for stimulation to condition the muscle. This was followed by 4 weeks of continuous stimulation. Thus, devices were implanted for 11 weeks. Before conditioning of the muscles was initiated, the partial muscle wrap pressure at rest was 42 +/- 27 cm H2O, which was higher than the incomplete alpha wrap resting pressure of 20 +/- 4 cm H2O. Stimulated partial flap pressure was 161 +/- 50 cm H2O, and stimulated modified alpha wrap pressures was 71 +/- 27 cm H2O. After conditioning with the modified alpha wrap, the resting and stimulated pressures were unchanged from before conditioning. Technical problems precluded collection of data during the conditioning period in dogs with partial flaps. During stimulation, the partial muscle wrap demonstrated marked deviation, whereas the modified alpha wrap had minimal urethral movement. Postmortem evaluation indicated no urethral stricture or fistula formation with either of the two types of wraps. The modified alpha wrap had several positive features. Advantages over the partial wrap were minimal resting pressures, reduced urethral mobility, and adequate sustained pressures during stimulation. Therefore, in contrast to the partial gracilis muscle wrap, aspects of the incomplete alpha wrap should be considered further for DUG.

Estimating detrusor pressure at home in pediatric patients with myelomeningocele.

PURPOSE: We evaluated a method of estimating detrusor pressure at home in patients with myelomeningocele who perform clean intermittent catheterization to empty the bladder. MATERIALS AND METHODS: Patients with myelomeningocele who perform clean intermittent catheterization underwent cystometry. At home they determined bladder pressure before draining a full bladder and after partial draining with the bladder almost empty. Home estimate of detrusor pressure was calculated using the formula, full bladder pressure - almost empty bladder pressure. RESULTS: A total of 4 boys and 5 girls with a mean age plus or minus standard deviation of 9.6+/-7.9 years who were enrolled in our study made 16.9+/-15.2 home bladder pressure and volume recordings weekly each during a mean of 5.8+/-4.3 months. Mean bladder capacity determined at home was significantly greater than cystometric capacity (354+/-185 versus 250+/-146 ml.). At a mean home and cystometric volume of 190+/-110 ml. full bladder pressure at home was not significantly different from cystometric vesical pressure (31.0+/-8.8 versus 27.5+/-7.5 cm. water). At a mean volume of 23+/-15 ml. mean home almost empty bladder pressure was not significantly different from cystometric abdominal pressure at full and almost empty volumes (14.1+/-5.5 versus 17.0+/-7.4 and 15.5+/-5.8 cm. water). Mean home estimate of detrusor pressure was not significantly different from cystometric detrusor pressure (17.0+/-6.3 versus 10.2+/-9.2 cm. water). CONCLUSIONS: Estimation of detrusor pressure at home is reliable and accurate in patients who perform clean intermittent catheterization. These pressure determinations may be used as a baseline for rapid identification of changes in bladder function.

Evaluation of a suture electrode for direct bladder stimulation in a lower motor neuron lesioned animal model.

The purpose of this study was to evaluate a "suture" type electrode for direct bladder stimulation in an animal model of a lower motor neuron lesion. During an initial surgery, five male cats were instrumented under anesthesia using multistranded, 316 LVM, stainless-steel, wire electrodes implanted on the bladder wall serosa above the trigone area. Electrodes were constructed with a needle attached to the end that was removed after suturing the electrode in place. Additional instrumentation included urinary bladder catheters (tubes) for pressure recording and filling, and hook type electrodes for leg and pelvic floor electromyography recording. Chronic bladder filling and stimulation studies were conducted in tethered animals three to four weeks following surgery. To test these electrodes in a spinal cord injury model, a lower motor neuron lesion was performed including the sacral cord and complete nerve roots at L6 and below. These animals were evaluated during weeks 3 and 10 after injury. Direct bladder stimulation induced active contractions and voiding both before and after spinal cord injury. Effective stimulation parameters consisted of 40 pulses per s, 300 micros to 1 ms pulse duration, a stimulation period from 3 to 4 s, and a stimulation current from 10 to 40 mA. Fluoroscopy revealed an open membranous urethra during stimulation and following stimulation. A small diameter penile urethra was observed to limit flow. Postmortem evaluation of the suture electrode revealed no abnormalities such as corrosion, migration into the bladder lumen or displacement. These findings indicate that suture electrodes are suitable and effective for short-term implantation in the lower motor neuron animal model.

Optimization of sacral ventral root stimulation following SCI: two case reports with six-month follow-up.

Sacral ventral root stimulation in conjunction with sacral dorsal rhizotomy has been effective in promoting voiding in individuals with upper-motor-neuron spinal cord injury. We report on two patients who had variable voiding responses to stimulation during the first six months after electrode implantation. We used videourodynamic records and daily voiding records to characterize their voiding difficulties. Different methods were used to improve voiding, including seating adjustments and changes in stimulation parameters. The first patient was unable to empty his bladder on a regular basis with stimulation using 24 pulses per sec stimulating frequency for the first two months after implantation. Voiding was substantially improved by using 35 pulses per sec. At the end of six months, he is regularly emptying his bladder with stimulation and is on an every-second-day bowel program. However, his bowel program has been irregular. The second patient had very good voiding when stimulation was applied in bed, but he had poor voiding with high residual volumes when sitting in his wheelchair. Voiding was improved when he used a wheelchair cushion that was cut out in the back or lifted his buttocks off the chair. These procedures appeared to reduce perineal pressures. This patient has bowel care on alternate days and his bowel care time has been reduced following implantation of the device. Neither of the patients experienced an erection with the device. Both patients feel positive about their implant experience.

Botulinum toxin injections for voiding dysfunction following SCI.

Botulinum toxin (BT) injections have been used successfully to treat spastic muscle conditions, including detrusor-sphincter dyssynergia (DSD) seen in spinal cord injury (SCI) patients. In our urology clinic, we used BT to treat three SCI patients who had voiding dysfunction, using a transperineal needle with electromyographic (EMG) monitoring. Two of the patients reported excellent results following the treatment. One patient, with whom the staff had difficulty doing intermittent catheterization (IC), improved significantly. The other patient had improved voiding with an external catheter and minimal urinary residual. The third patient had no improvement of leg spasms with his voiding dysfunction and required a sphincterotomy. Although patients may need repeat injections, BT is minimally invasive and easy to administer with no side effects. Overall, BT injection is an excellent method of managing voiding in SCI patients, especially those on continuous external catheters and with IC management who refuse or are not good candidates for surgery.

The use of hematological effects in the development of minimal risk levels.

The Agency for Toxic Substances and Disease Registry (ATSDR) derives minimal risk levels (MRLs) to assist in evaluating risk of adverse health effects in individuals exposed to hazardous substances. MRLs are derived from published values identifying no-observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) in animal or human studies. The most sensitive end points are used. To date, 4 inhalation MRLs and 13 oral MRLs have been derived from hematological end points for 12 substances. This paper provides a brief overview of the hematological system, examples of hematological end points, and the MRL for substances with hematological end points.

Home bladder pressure monitoring in children with myelomeningocele.

PURPOSE: We evaluate a pressure gauge used at home for patients with myelomeningocele on clean intermittent catheterization to provide a system for inexpensive frequent monitoring of bladder pressures. MATERIALS AND METHODS: Subjects with myelomeningocele using clean intermittent catheterization underwent cystometry in the laboratory. At home they obtained weekly volumes and bladder pressures before and after emptying. Home estimate of detrusor pressure was defined as full bladder pressure minus empty bladder pressure. Medication changes, subject position and urinary tract symptoms were noted. RESULTS: A total of 11 subjects 10.5+/-7.3 years old have been enrolled and have made 16.7+/-12.6 weekly home bladder pressure and volume recordings in 4.7+/-3.1 months. Bladder capacities measured at home were 132+/-47% of cystometric capacities. At volumes of data overlap home full pressures (31+/-10 cm. water) were not statistically different from cystometric vesical pressures (25+/-9 cm. water). Home empty pressures (7+/-4 cm. water) were similar to cystometric abdominal pressures (14+/-8 cm. water). Home estimates of detrusor pressures (23+/-7 cm. water) magnified differences in full and empty pressures, and were significantly greater than cystometric detrusor pressures (11+/-11 cm. water). In 2 subjects significant increases in home full pressures occurred, which were associated with cessation of anticholinergic medication and infection. CONCLUSIONS: Home monitoring of bladder pressure is a simple, inexpensive and accurate method of obtaining frequent bladder pressures in patients with myelomeningocele. These pressures are consistent over a large range of volumes and times, and could potentially be used to identify quickly changes in patient condition.

Home monitoring of bladder pressure and volume in individuals with spinal cord injury and multiple sclerosis.

Individuals with spinal cord injury and multiple sclerosis are at high risk for developing kidney dysfunction due to high bladder pressures. We have developed a device for frequent monitoring of bladder pressures at home in those patients who use intermittent catheterization to empty their bladders. Of eight subjects enrolled in the study, only five conducted home recording of pressure. Vesical and abdominal pressures measured at home were significantly lower than clinical cystometric pressures. However, subtracted detrusor pressures obtained from home records and cystometric records were not significantly different. The home detrusor pressures were consistent over a large time and volume range. Therefore, the home monitoring method could be used to establish a normal range of bladder pressures at home and to rapidly identify high bladder pressures in advance of upper urinary tract deterioration.

Urodynamic verification of noninvasive back-pressure recordings from the urinary bladder.

Obstructive voiding is best evaluated with urodynamics, especially simultaneous measurement of bladder-pressure and urine flow rates. As an alternative to catheterization for urodynamics, noninvasive back-pressure methods using an external condom system have been introduced. This device uses one side tube in the condom for pressure recording and an outlet tube that is clamped for short periods of time during voiding. However, there have been problems with accurate back-pressure recording, including leaking, clamping techniques, hydrostatic pressures associated with pressure recording below the level of the symphysis pubis, and assessment of back pressures in relation to bladder and detrusor pressures. To address these issues, we have modified the condom for passing a catheter into the urethra for simultaneous direct bladder and back-pressure recording. The clamping device on the outlet tube also has been modified to produce back flushing of urine in addition to clamping. Hydrostatic issues have been addressed by making pressure recordings at the level of the symphysis pubis. Seven patients with obstructive symptoms were evaluated using these new devices. Back pressures were not statistically different than detrusor pressures recorded with a urethral catheter. Thus, the modifications have improved back-pressure recording techniques. The use of noninvasive back-pressure recording may be an important adjunct in the evaluation of obstructive uropathy.

Use of the HIV-1 protease for excision of growth-hormone-releasing factor from synthetic and recombinant peptide precursors.

An autolysis-resistant mutant of the HIV-I protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors. The N-terminal four amino acids in two selected model GRF analogues, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates. A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4'. A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe-1-Tyr1 bond (... RQVF-decreases-YIDA ...) to release GRF32. However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success. By random mutagenesis in a propeptide segment, [MGQSVAQVF]-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E. coli and was effectively processed by the HIV-I protease. A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.

Short-term bladder-wall response to implantation of microstimulators.

This study examined the histological response of the bladder wall serosa to the implantation of wireless microstimulators secured with a single polypropylene suture. Two to three microstimulators were implanted in each of three casts for an eight week implantation period to allow sufficient time for a bladder-wall injury-response to develop. Gross observation revealed encapsulation of the microstimulators with no perforation to the bladder lumen or migration from the bladder wall. Histological evaluation confirmed that all the microstimutators were encapsulated with a thin connective tissue sheath and a thickened subserosal layer. There was no remarkable difference in tissue morphology compared with normal bladder wall sections for five of seven stimulators. Two microstimulators in one cat revealed a moderate to severe inflammatory response confined to a small area around the stimulator. In a second cat, a suture extended through the bladder wall. The microstimulators were observed with a scanning electron microscope after explantation. The electrode surfaces, bonding interface between silicon and glass and insulating films that were exposed to biological fluids were carefully inspected. All these observations indicate that the glass capsule reliably protected the sealed cavity of the microstimulators from moisture. These results indicate the microstimulator should be considered for further studies such as effects of stimulation and long-term implantation.

Direct bladder stimulation with suture electrodes promotes voiding in a spinal animal model: a technical report.

To determine the efficacy of a new electrode for direct bladder stimulation, five male cats were instrumented during anesthesia. Multistranded, 316LVM, stainless-steel, wire electrodes were implanted on the bladder wall serosa above the trigone area. The electrodes were made with a needle attached to the end that was cut off after suturing the electrode in place. Additional instrumentation included tubes for pressure recording and filling, and hook electrodes for leg and pelvic floor EMG recording. Bladder filling and stimulation studies were conducted in tethered animals 1 to 2 weeks following recovery. Chronic studies were conducted following recovery in tethered animals. To test these electrodes in a spinal cord injury (SCI) model, a T-1 level complete lesion was performed on the above instrumented animals. Spinal animals had successful direct bladder stimulation that induced active contractions and voiding both before and after SCI, but voiding rates were higher more than 2 weeks after SCI and at larger initial bladder volumes. Optimum stimulation parameters consisted of 40 pulses per second, 300 microseconds to 1 ms pulse duration, a stimulation period of 3 to 4 s, and 10 to 40 mA. Urethral resistance, indicated by a urethral function measure, showed that stimulation had no adverse effect on urethral function, and fluoroscopy showed an open membranous urethra during stimulation and voiding. The cat has a small penile urethra that is the flow rate controlling zone. The suture electrode did not corrode, erode into the bladder, or become dislodged, and appears suitable for chronic implantation.

Identification of signal-induced IkappaB-alpha kinases in human endothelial cells.

Activation of the nuclear transcription factor-kappaB is an early event in endothelial activation. NF-kappaB activation is regulated by the inducible phosphorylation and subsequent degradation of the inhibitory subunit IkappaB-alpha. We identified two discrete kinases of approximately 36 and 41 kDa in the cytoplasm of human umbilical vein endothelial cells that specifically bind to and phosphorylate the IkappaB-alpha subunit. IkappaB-alpha kinase activity is transiently elevated following treatment with either tumor necrosis factor alpha, interleukin-1beta, or bacterial lipopolysaccharides and precedes activation of either mitogen-activated kinase or Jun kinase. Furthermore, activation of the IkappaB-alpha kinases precedes both the appearance of hyperphosphorylated IkappaB-alpha and its subsequent degradation, as well as the translocation of NF-kappaB to the nucleus. Deletion mutagenesis of the IkappaB-alpha polypeptide revealed that these kinases bind in or around the ankyrin repeat domains and phosphorylate residues within the C terminus. These kinases, however, were not identical to casein kinase II and displayed a pharmacologic profile distinct from other known kinases. These kinases may represent components of a signal transduction pathway regulating IkappaB-alpha levels in vascular endothelium.

Preliminary noninvasive back-pressure recordings of bladder pressure.

Obstructive voiding is best evaluated with urodynamics, including bladder pressure and urine flow rates. Until recently, the recording of bladder pressure required the use of a urethral catheter. In preliminary observations, a noninvasive back-pressure method using an external condom catheter has been introduced to determine bladder pressure. This device uses a side tube for pressure recording and an outlet tube that is clamped for short periods of time. We have investigated design criteria for back-pressure recording techniques. In the laboratory setting using a plastic model, we determined that a low compliance condom is needed. In addition, a back flow of fluid during the clamping procedure helps to obtain quick back pressures and facilitates evaluation of pressure when low flow rates are present. These modified condom devices were evaluated in four male subjects. Back pressures were not statistically different than bladder pressures recorded with a urethral catheter. The use of back pressures in the evaluation for obstructive uropathy can be enhanced by using a pressure and flow nomogram.