Prospective evaluation of 4-D contrast-enhanced-ultrasound (CEUS) imaging in bladder tumors.

PURPOSE: The evaluation of the potential clinical benefit of four-dimensional ultrasound (4D-US) in the assessment of bladder cancer (BC). MATERIAL AND METHODS: 20 patients with indication for cystoscopy for suspicion of bladder cancer were prospectively included in this study. All patients underwent two-dimensional ultrasound (2D-US), contrast enhanced ultrasound (CEUS) and real-time four-dimensional ultrasound (4D-US). All acquisitions were compared to each other in regard to image quality. This assessment was done using a 6 point scale (1 = best). All patients underwent subsequently cystoscopy with resection of the tumor (TURB), due a histopathological analysis was possible. RESULTS: All examinations were performed successfully and no patient had to be excluded from the study. Patients acceptance of 4D-US was consistently good. No adverse events occurred. Image quality of real time 4D-US (score: 1.27±0.46) was significantly superior (p < 0.001) to both, conventional 2D-US (score: 2.33±0.62) and also to 2D-CEUS (score: 2.00±0.53). In terms of tumor detection no superiority was evident for 4D-US compared to 2D-US or in utilization of CEUS (sensitivity = 0.89; specificity = 1.00; positive predictive value = 1.00; negative predictive value = 0.50; AUC = 0.944; (95% CI: 07.43-0.998)). CONCLUSION: The assessment of bladder cancer using real time 4D-US is feasible and improves the image quality and therefore also the precise anatomical consistency of intravesical tumor masses.

[Current controversies in the treatment of localized prostate cancer]. / Aktuelle Kontroversen in der Therapie des lokal begrenzten Prostatakarzinoms.

In the prostate-specific antigen (PSA) era, most prostate cancers (PCa) are diagnosed in a localized stage and a plethora of therapeutic options are warranted in different clinical settings and disease stages of localized PCa. In the current narrative review, we give an overview of the current controversies in the therapeutic landscape of localized PCa and focus on organ-sparing approaches, percutaneous radiotherapy, brachytherapy as well as retropubic and robot-assisted prostatectomy by summarizing studies that have been published within the last two years.

The viability of chondrocytes after an in vivo injection of local anaesthetic and/or corticosteroid: a laboratory study using a rat model.

This in vivo controlled laboratory study was performed to evaluate various intra-articular clinical injection regimes that might be less toxic than some in vitro studies suggest. We hypothesised that low-concentration, preservative-free, pH-balanced agents would be less toxic than high-concentration non-pH-balanced agents with preservatives, and that injections of individual agents are less toxic than combined injections. The left knees of 12- to 13-week-old Sprague-Dawley rats were injected once with eight different single agents, including low and high concentrations of ropivacaine and triamcinolone, alone and in combination, as well as negative and positive controls. The rats were killed at one week or five months, and live-dead staining was performed to quantify the death of chondrocytes. All injections except pH-balanced 0.2% ropivacaine combined with preservative-free 1 mg/ml triamcinolone acetonide resulted in statistically significant decreases in chondrocyte viability, compared with control knees, after one week and five months (p < 0.001). After one week there was no significant difference in viability between 0.2% and 0.5% ropivacaine; however, 4 mg/ml triamcinolone resulted in a lower viability than 1 mg/ml triamcinolone. Although many agents commonly injected into joints are chondrotoxic, in this in vivo study diluting preservative-free 10 mg/ml triamcinolone 1:9 in 0.2% pH-balanced ropivacaine resulted in low toxicity.

The influences of sex and posture on joint energetics during drop landings.

Previous observations suggest that females utilize a more erect initial landing posture than males with sex differences in landing posture possibly related to sex-specific energy absorption (EA) strategies. However, sex-specific EA strategies have only been observed when accompanied by sex differences in initial landing posture. This study (a) investigated the potential existence of sex-specific EA strategies; and (b) determined the influences of sex and initial landing posture on the biomechanical determinants of EA. The landing biomechanics of 80 subjects were recorded during drop landings in Preferred, Flexed, and Erect conditions. No sex differences in joint EA were identified after controlling for initial landing posture. Males and females exhibited greater ankle EA during Erect vs Flexed landings with this increase driven by 12% greater ankle velocity, but no change in ankle extensor moment. No differences in hip and knee EA were observed between conditions. However, to achieve similar knee EA, subjects used 7% greater mean knee extensor moment but 9% less knee angular velocity during Flexed landings. The results suggest that sex-specific EA strategies do not exist, and that the magnitude of knee joint EA can be maintained by modulating the relative contributions of joint moment and angular velocity to EA.

[Etiology and pathophysiology of benign prostate hyperplasia]. / Ätiologie und Pathophysiologie der benignen Prostatahyperplasie.

The pathogenesis of benign prostate hyperplasia (BPH) is still unclear. It is a common disease affecting exclusively humans in its full clinical appearance. There is a broad variety of possible underlying mechanisms which most likely interact in the pathogenesis of the disease: inflammatory processes taking place predominantly in the stroma and inducing proliferation of all tissues within the transitional zone, an imbalance of androgens and estrogens and their receptors, hyperinsulinemia and hypercholesterolemia (metabolic syndrome) as direct promoters of glandular growth and autosomal dominant inheritance. The detrusor muscle responds to the increased outflow resistance with muscular hypertrophy. Decreased compliance of the bladder wall results in voiding difficulties while electric instability of the hypertrophied detrusor muscle and increased recruiting of otherwise silent afferent fibres cause storage symptoms.

Comparison of the mechanical properties of different tension band materials and suture techniques.

OBJECTIVES: This study determined the tensile properties of 18-gauge stainless steel wire, 5-mm woven polyester (Mersilene) tape, and multiple loop configurations of No. 5 braided polyester suture (Ethibond). DESIGN: Mechanical property testing. INTERVENTION: Single loops of stainless steel wire, Mersilene, and Ethibond were tested to determine their mechanical properties. Ethibond was tested with different numbers of loops and different knot configurations. MAIN OUTCOME MEASURES: Stiffness, load at failure, and elongation at failure. RESULTS: One loop of Mersilene and two loops of Ethibond had similar loads at failure, but the load at failure was significantly higher for stainless steel wire. Four loops of Ethibond withstood a similar failure load to stainless steel wire, but the failure load of the Ethibond suture was greater than the yield load of stainless steel wire. Stainless steel wire had a higher stiffness than both Ethibond and Mersilene. No difference was found in the failure load between different Ethibond knot configurations. The individually tied suture configuration resulted in a higher stiffness than the single-knot configuration. The elongation at failure was not statistically different among the different knot configurations and materials, with the exception of Mersilene tape. Mersilene tape demonstrated a significant increase in elongation at failure as compared to the other materials and knot configurations. CONCLUSION: It appears that multiple loops of Ethibond can substitute for stainless steel wire in situations where a compliant repair is suitable (support of a patellar tendon repair), but may not be satisfactory for rigid fixation (tension band fixation of a fracture). There appears to be no significant difference in strength but a small decrease in stiffness between tying multiple suture loops in one knot as opposed to individual knots.

A cyclooxygenase-2 inhibitor impairs ligament healing in the rat.

Celecoxib was the first of a new class of nonsteroidal antiinflammatory drugs, the cyclooxygenase-2 (COX-2) specific inhibitors, marketed as having the same antiinflammatory efficacy as other nonsteroidal antiinflammatory drugs without their increased risk of gastrointestinal ulceration. Among the widest uses of nonsteroidal antiinflammatory drugs is in the treatment of acute soft tissue injuries. Although the benefits of celecoxib have been shown when used for rheumatoid arthritis and osteoarthritis, we are unaware of any studies concerning its effect on soft tissues. We used the surgically incised medial collateral ligament of male Sprague-Dawley rats as an experimental model for acute ligament injuries to investigate the effects of celecoxib on ligament healing. Fifty rats underwent surgical transection of the right medial collateral ligament. Postoperatively, half were given celecoxib for the first 6 days of recovery, the other half were not. The animals were sacrificed 14 days after the operation, and both the injured and uninjured medial collateral ligaments were mechanically tested to failure in tension. Celecoxib-treated/injured ligaments were found to have a 32% lower load to failure than untreated/injured ligaments. The results of this study do not support use of cyclooxygenase-2 specific inhibitors in the treatment of ligament injuries.

The effect of NKISK on tendon in an in vivo model.

The mechanism through which ligaments and tendons change length during growth and contracture is unclear. It has been hypothesized that there is a reversible "interfibrillar bond" that, when broken, allows the sliding of collagen fibrils past one another during length changes. The pentapeptide NKISK has been reported to inhibit the binding of decorin to fibronectin. This study was designed to evaluate the effect of NKISK in an in vivo model. Male Sprague-Dawley rats were divided into three groups (n = 9, 9 and 14, respectively). The left patellar tendon was injected with 1.0 ml of NKISK (Group 1 = 1.0 mM, Groups 2 and 3 = 5.0 mM). The contralateral/control limb was injected with carrier. Group 1 was sacrificed after three, Group 2 after four and Group 3 after seven daily injections. The patellar tendon lengths were measured in all groups with comparisons made to the contralateral control limb. NKISK injection resulted in a significant increase in length in Group 2 (3.14% +/- 2.04, P = 0.002) and in Group 3 (6.12% +/- 3.84, P < 0.001). Biomechanical testing of Group 3 showed no differences in maximum load, ultimate strength, structural stiffness, or elastic modulus of the treated tendons but did demonstrate a statistically significant decrease in the displacement and strain at maximum load in the NKISK-treated tendons. This study demonstrates that inhibition of decorin/fibronectin binding by NKISK results in tendon lengthening in an in vivo setting as noted by a progressive increase in the length of the patellar tendon.

Computer simulation of trabecular remodeling using a simplified structural model.

A simplified three-dimensional simulation of trabecular bone remodeling has been developed. The model utilizes 441 planar structural units to represent approximately 50 mm3 of initial bone volume with 199 basic multicellular units (BMUs). The simulation takes into account trabecular perforation in the structural model. The cases of male bone remodeling with no menopause and female bone remodeling with menopause are examined from the period of simulated age 25-80 years. Menopause is arbitrarily started at age 45 and extends for 7.5 years. Zero-, first-, and second-order BMU activation responses are employed to examine how the bone would be affected by the method of increase of BMU activation during menopause. At age 80, the female bone remodeling simulation produced a bone volume loss of approximately 49% for all three activation responses. This compared to a 38% bone volume loss for the case of no menopause. For the menopause simulations, an average of about 40% of the total bone loss was due to perforation.

Gap junctions regulate responses of tendon cells ex vivo to mechanical loading.

Avian digital flexor tendons were used with a device to apply load ex vivo to study the effects on deoxyribonucleic acid and collagen synthesis when cell to cell communication is blocked. Flexor digitorum profundus tendons from the middle toe of 52-day-old White Leghorn chickens were excised and used as nonloaded controls, or clamped in the jaws of a displacement controlled tissue loading device and mechanically loaded for 3 days at a nominal 0.65% elongation at 1 Hz for 8 hours per day with 16 hours rest. Tendon samples were radiolabeled during the last 16 hours with 3H-thymidine to monitor deoxyribonucleic acid synthesis or with 3H-proline to radiolabel newly synthesized collagen. Cyclic loading of whole avian flexor tendons stimulated deoxyribonucleic acid and collagen synthesis, which could be blocked with octanol, a reversible gap junction blocker. Cells from human digital flexor tendon were used to populate a rectangular, three-dimensional, porous, polyester foam that could be deformed cyclically in vitro. Together, these results support the hypothesis that tendon cells must communicate to sustain growth and matrix expression and that an engineered three-dimensional construct can be used to study responses to mechanical load in vitro.

Enhancement of mdr2 gene transcription mediates the biliary transfer of phosphatidylcholine supplied by an increased biosynthesis in the pravastatin-treated rat.

An increase of biliary lipid secretion is known to occur in the rat under sustained administration of statin-type 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors. The present study has addressed critical mechanisms of hepatic lipid synthesis and phosphatidylcholine (PC) biliary transport in the rat fed with a 0.075% pravastatin diet for 3 weeks. After treatment, biliary secretion of PC and cholesterol increased to 233% and 249% of controls, while that of bile salts was unchanged. Activity of cytidylyltransferase (CT), a major regulatory enzyme in the CDP-choline pathway of PC synthesis, was raised in both microsomal and cytosolic fractions (226% and 150% of controls), and there was an increase to 187% in the mass of active enzyme as determined by Western blot of microsomal protein using an antibody specific to CT. Cytosolic activity of choline kinase, another enzyme of the CDP-choline pathway, also increased to 175% of controls. In addition, there was an over eightfold increase in the HMG CoA reductase activity and mRNA. Thus, an increased PC and cholesterol synthetic supply to hepatocytes appeared as a basic mechanism for the biliary hypersecretion of these lipids. Notwithstanding the increased synthesis, hepatic PC content was unchanged, suggesting an enhanced transfer of this lipid into bile. Indeed, there was a sevenfold increase of multidrug resistance gene 2 (mdr2) gene mRNA coding for a main PC canalicular translocase. Thus, hypersecretion of biliary PC in the model studied can be explained by an up-regulation of mdr2 gene transcription and its P-glycoprotein product mediating the biliary transfer of PC supplied by an increased biosynthesis.

Assessment of the directional elastic moduli of ewe vertebral cancellous bone by vibrational testing.

The ovariectomized ewe is being used as an animal model for postmenopausal osteoporosis. Data on the mechanical properties of ewe vertebral cancellous bone is needed to assess its effectiveness as a model for vertebral osteoporosis. This study utilized traditional compression testing and a novel nondestructive vibrational testing method to assess the directional mechanical properties of ewe vertebral cancellous bone. Composition and density properties were also assessed. It was hypothesized that vibrational testing would have utility in that it would allow for the anisotropic stiffness of cancellous bone to be assessed nondestructively. The present study has found that ewe vertebral cancellous bone has similar physical and mechanical properties to humans. The vibrational testing method described was able to nondestructively provide a valid measure of stiffness that was correlated with stiffness estimates from traditional compression testing. Furthermore, the stiffness measure from the vibration test was found to be sensitive to the architecture of cancellous bone. These results suggest the promise of this testing method for the nondestructive mechanical assessment of skeletal tissue.

Comparison of tricortical screw fixation versus a modified suture construct for fixation of ankle syndesmosis injury: a biomechanical study.

OBJECTIVE: To assess the viability of using a modified flexible suture implant instead of a tricortical screw for fixation of ankle syndesmosis failures. DESIGN: Randomized biomechanical study. SETTING: Orthopaedic Research Laboratories at the University of North Carolina at Chapel Hill. INTERVENTION: Formalin-preserved cadaveric legs were used in pairs. Two holes, 2.5 millimeters in diameter and 7.0 to 10.0 millimeters apart horizontally, were drilled through the fibula and tibia 2.0 centimeters above the tibial plafond. Two strands of Number 5 suture were passed through the holes and tied. Similarly, a 3.5-millimeter tricortical screw was placed on the opposite leg of each pair at 2.0 centimeters. The ankles were tested to failure. This process was repeated at 5.0 centimeters above the tibial plafond. MAIN OUTCOME MEASUREMENTS: Maximum load and displacement at failure of the suture construct at 2.0 centimeters and at 5.0 centimeters were compared with a tricortical screw at 2.0 centimeters and at 5.0 centimeters. RESULTS: There was no significant difference in strength or displacement between the flexible suture implant and the tricortical screw at either 2.0 centimeters or 5.0 centimeters. The fixations at 5.0 centimeters had significantly increased holding strength over fixations at 2.0 centimeters, and the fixations had significantly greater displacement at 2.0 centimeters than at 5.0 centimeters. CONCLUSION: This study confirms that flexible syndesmosis repair is a viable option for internal fixation of ankle mortise instability due to syndesmosis rupture.

Studies on the regulation of CTP:phosphocholine cytidylyltransferase using permeabilized HEP G2 cells: evidence that both active and inactive enzyme are membrane-bound.

To obtain more insight into the mechanisms regulating CTP:phosphocholine cytidylyltransferase (CT), we determined the effect of oleate treatment on the rate of CT release from permeabilized Hep G2 cells and the distribution of the CT remaining in the permeabilized cells. When we permeabilized untreated cells in pH 7.5 buffer containing 0.15 M KCl, the rate of CT release was much slower than the release of lactate dehydrogenase. Oleate treatment caused a further decrease in CT release from cells. In untreated cells, 70-80% of the CT remaining in cells 10 min after permeabilization was recovered as soluble CT. Oleate treatment increased the amount of bound CT but over 50% of the CT in cells 10 min after permeabilization was recovered as soluble CT. In both control and oleate-treated cells, the increase in CT release with time correlated with a decrease in the amount of CT recovered from permeabilized cells as soluble CT. These results suggested that CT existed in a form that was not immediately available for release from permeabilized cells, but was recovered in the soluble fraction after cell disruption. When cells were permeabilized in 10 mM imidazole-20% glycerol-5 mM Mg2+ pH 6.5, over 80% of CT in control and over 90% of CT in oleate-treated cells was recovered bound to the particulate fraction. Essentially no CT was released from the cells. The recovery of CT in the particulate fraction required Mg2+ to be present when permeabilization was initiated. The addition of Mg2+, after cells were disrupted, did not increase CT in the particulate fraction. In untreated cells, 50% of bound CT was active. Oleate treatment increased the amount of active CT in the particulate fraction to over 70% of total. About 50% of particulate CT in untreated cells but only 15% in oleate-treated cells was extracted with 0.15 M KCl. Inactive CT was preferentially extracted by KCl. The bound CT was recovered in isolated nuclei. Overall, the results suggested that both inactive and active CT are bound to nuclear membranes, and that the activation of CT involves conversion of CT loosely bound to membrane to a form more tightly bound to membranes perhaps by hydrophobic interaction with phospholipids. This model does not involve translocation from a soluble pool.

Cytidylyltransferase-binding protein is identical to transcytosis-associated protein (TAP/p115) and enhances the lipid activation of cytidylyltransferase.

We previously identified a protein from rat liver that binds CTP:phosphocholine cytidylyltransferase (CT). We have now purified this protein (cytidylyltransferase-binding protein (CTBP)) from rat liver. The purification involved precipitation at pH 5 and extraction of the precipitate with buffer, followed by sequential chromatography on DEAE-Sepharose and butyl-agarose. Final purification was accomplished by either preparative electrophoresis or hydroxylapatite chromatography. Amino acid sequences from six peptides derived from pure CTBP matched sequences in transcytosis-associated protein (TAP) with 98% identity. Thus, CTBP was positively identified to be TAP. Purified CTBP increased the activity of purified CT measured with phosphatidylcholine (PC)/oleic acid. In the absence of PC/oleic acid, CTBP did not stimulate CT activity. Dilution of CT to reduce the Triton X-100 concentration produced a loss of CT activity. The lost activity was recovered by the addition of CTBP plus PC/oleic acid to the assay, but not by the addition of either PC/oleic acid or CTBP alone. Removal of CTBP from purified preparations by immunoprecipitation with CTBP antibodies eliminated the activation of CT. Both CT and CTBP were shown to bind to PC/oleic acid liposomes. The formation of complexes between CT and CTBP in the absence of PC/oleic acid liposomes could not be demonstrated. These results suggest that CTBP functions to modify the interaction of CT with PC/oleic acid liposomes, resulting in an increase in the catalytic activity perhaps by the formation of a ternary complex between CT, CTBP, and lipid. Overall, these results suggest that CTBP (TAP) may function to coordinate the biosynthesis of phosphatidylcholine with vesicle transport.

Fatty acids promote the formation of complexes between choline-phosphate cytidylyltransferase and cytidylyltransferase binding protein.

We previously identified a 112-kDa protein (CTBP) that binds CTP:choline-phosphate cytidylyltransferase (CT) (D.A. Feldman and P.A. Weinhold, 1993, J. Biol. Chem. 268, 3127-3135). In this study we show that fatty acids promote the binding of cytidylyltransferase to CTBP. Gel filtration chromatography separated CTBP from CT in liver cytosol. CTBP was eluted slightly slower than the thyroglobulin standard. The addition of oleate to cytosol followed by incubation at 37 degrees C resulted in the formation of aggregates containing both CT and CTBP. The aggregates eluted in the void volume of the gel filtration column, sedimented to the bottom of glycerol density gradients, and precipitated at concentrations of polyethylene glycol lower than required to precipitate CT and CTBP in untreated cytosol. Immunoprecipitation by CT antibodies of both CT and CTBP in aggregate preparations provided further evidence for the formation of CT-CTBP complexes. A smaller CT-CTBP complex was isolated by glycerol density centrifugation from cytosol incubated with oleate at 4 degrees C. Overall, these results suggest that oleate promotes the binding of CT to CTBP. The results suggest that a dissociable complex is formed at 4 degrees C in the presence of oleate which is polymerized by incubation at 37 degrees C to a large aggregate that is more resistant to dissociation. Complex formation at 37 and 4 degrees C was dose dependent at oleate concentrations between 50 and 200 microM. Complex formation at 4 degrees C was specifically promoted by long-chain, unsaturated fatty acids. These results suggest that CTBP may be involved in the fatty acid-induced translocation of cytidylyltransferase.

The significance of transient changes in trabecular bone remodeling activation.

After menopause, bone turnover is increased due to an increase in the activation of bone remodeling basic multicellular units (BMUs). The importance of changes in BMU activation to bone histomorphometry data and bone volume has not received adequate attention by many skeletal researchers. Therefore, the influence of BMU activation on the above parameters was modeled by way of computer simulation, and the results were compared to data from published post-menopausal bone loss studies. Using control theory concepts, the increase in BMU activation after menopause was modeled as a zero-, first-, or underdamped (oscillatory) second-order transient BMU activation response to the step input: the decline in estrogen. A computer simulation was developed to model the influence of these three transient BMU activation responses on quantitative histological surface parameters and bone volume. The transient BMU activation responses doubled the number of active BMUs. All three types of transient BMU activation responses produced a rapid 5% decline in bone volume due to increased remodeling space. Oscillations in bone volume and histologic surface parameters over time, similar in nature to those seen in studies of ovariectomized animals, were predicted by the simulation for the oscillatory activation response examined, underdamped second order. An oscillatory BMU activation response may explain some of the transient events during menopause. The increased coherence of BMUs created by such a response may increase the likelihood of trabecular perforations. the inherent nature of an oscillatory activation response may cause its detection to be overlooked and bone remodeling data to be misinterpreted.(ABSTRACT TRUNCATED AT 250 WORDS)

Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro.

Cells in the body are constantly subjected to cyclic mechanical deformation involving tension, compression, or shear strain or all three. A mechanical loading system which deforms cultured cells in vitro was analyzed in order to quantify the deformation or strain to which the cells are subjected. The dynamic system utilizes vacuum pressure to deform a circular silicone rubber substrate on which cells are cultured. These thick circular growth surfaces or plates are formed in the bottoms of the wells of 6-well culture plates. An axisymmetric model was formulated and analyzed using rectangular hyperelastic elements in a finite element analysis (FEA) software package. The thick circular plate has some disadvantages such as difficulty in observing cells and a nonhomogeneous strain profile which is maximum at the periphery and minimal at the center. A thinner circular surface (a thin plate) was also investigated in order to provide a more homogeneous strain profile. The radial strain on the thick circular plate, as determined by FEA, was nonlinear with a peak strain value of 0.30 (vacuum pressure of 22 kPa) about three-quarters of the distance from the center to the edge. In contrast, the radial strain of the thin circular plate was moderately constant across the surface. The circumferential strain for both of these models was less than the radial strain except for the center where they are equal. Avian tendon cells were cultured on the surface of a thick plate and exposed to cyclic strains for 24 h at a rate of 0.17 Hz and observed for cellular alignment.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of CTP: phosphocholine cytidylyltransferase in HepG2 cells: effect of choline depletion on phosphorylation, translocation and phosphatidylcholine levels.

We studied the effect of choline depletion on the biosynthesis of phosphatidylcholine (PC) and the distribution and phosphorylation of cytidylyltransferase (CT) in HepG2 cells. Phosphocholine concentrations decreased within 24 h of choline depletion to values less than 2% of controls. The incorporation of [3H]glycerol into PC was reduced in choline-depleted (CD) cells. The apparent turnover of PC was similar in CD and choline-supplemented (CS) cells (T1/2 = 20 h). The methylation pathway for PC synthesis increased nearly 10-fold in CD cells. Cell growth was similar in CD and CS cells. Over 95% of CT activity in CS cells was in the soluble pool. Choline depletion resulted in a progressive decrease in CT activity and immunodetected enzyme in the soluble pool and a corresponding increase in membrane CT over a 48-h period. Choline supplementation of CD cells caused a rapid release of membrane CT (complete release by 3 h). Two phosphorylated forms of CT were identified. One form contained a higher level of phosphorylation (HPCT) than the other form (LPCT). HPCT migrated slightly slower than LPCT on SDS gels. CD cells contained only LPCT in both soluble and membrane pools. CS cells contained only HPCT. During choline depletion PC content decreased nearly 20% but CT binding did not occur until LPCT was generated in cytosol. Conversely, choline supplementation released LPCT into cytosol and HPCT was formed only after the release. We conclude that both the induction of binding sites, perhaps by depletion of PC and dephosphorylation of HPCT to LPCT, are required for CT translocation to membranes. The release of CT from membranes is initiated by changes in membrane binding sites followed by trapping of the CT in the soluble pool by phosphorylation of LPCT to HPCT.

Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase.

The soluble form of CTP:phosphocholine cytidylyltransferase, which has previously been assumed to be cytosolic, has been localized to the nucleus of several cell types. Indirect immunofluorescence microscopy indicated a nuclear location in HepG2, NIH-3T3, and L-cells. A comparison of the fluorescence pattern of wild-type CHO cells with a cytidylyltransferase-deficient mutant provided genetic evidence that cytidylyltransferase is nuclear in CHO cells. The enzyme is also predominantly nuclear in rat liver, as revealed by staining frozen sections of that tissue. When L-cells were fractionated by enucleation, over 95% of cytidylyltransferase activity was found in the nuclear fraction, providing biochemical evidence for a nuclear location in these cells. In light of the demonstration that the membrane-bound cytidylyltransferase in CHO cells is associated with the nuclear envelope (Watkins, J. D., and Kent, C. (1992) J. Biol. Chem. 267, 5686-5692), these results suggest that this enzyme is predominantly an intranuclear enzyme.