T1-weighted parenchyma attenuated inversion recovery: A novel sequence that improves contrast ratio of enhancing brain lesions.

PURPOSE: The purpose of this study was to develop and test a parenchyma attenuated T1-weighted inversion recovery MR sequence (PAIR) that increases the contrast between enhancing and non-enhancing tissues in the brain and to compare the contrast ratio of enhancing brain tumors on this sequence compared to spin echo magnetization transfer (SEMT). PATIENTS AND METHODS: PAIR sequence parameters were developed to reduce signal from gray matter (GM), white matter (WM), and cerebrospinal fluid (CSF) in a healthy adult volunteer. Forty-one patients (17 men and 24 women) with a mean age of 55±13 (SD) years (range: 21-78years) with known or suspected brain tumors underwent PAIR and SEMT imaging after intravenous administration of gadobenate dimeglumine. In patients with confirmed tumors, PAIR and SEMT images were compared for contrast ratio of tumor-to-WM, tumor-to-GM, and tumor-to-CSF. RESULTS: A total of 23 enhancing neoplastic lesions were found in 14/41 patients. All tumors were visualized on both contrast enhanced PAIR and SEMT images. PAIR images showed a 2.5 fold increase in maximum tumor-to-GM contrast ratio (P<0.0001), a 1.4 fold increase in maximum tumor-to-WM contrast ratio (P=0.0007) and a 5-fold increase in maximum tumor-to-CSF contrast ratio (P<0.0001). CONCLUSION: PAIR provides improved lesion-to-background contrast ratio compared to SEMT and may be useful as an added sequence in tumor evaluation.

Clinical and demographic characteristics predictive of treatment outcomes for certolizumab pegol in moderate to severe Crohn's disease: analyses from the 7-year PRECiSE 3 study.

BACKGROUND: Clinical factors were previously identified as predictors of short-term treatment efficacy in Crohn's disease (CD). The PRECiSE 3 (P3) 7-year trial provides an opportunity to study predictors of short- and long-term clinical remission among CD patients treated with certolizumab pegol (CZP). AIM: To identify factors that influence long-term remission of CD with CZP treatment. METHODS: Patients who had completed placebo-controlled studies (PRECiSE 1/PRECiSE 2, P1/P2) enrolled in P3 and received open-label CZP 400 mg every 4 weeks up to 7 years. Baseline predictors included, but were not limited to, smoking status, disease duration, prior inflammatory bowel disease (IBD) surgery, Harvey-Bradshaw Index (HBI), albumin, haematocrit and CZP exposure; association with time to initial remission (HBI ≤4) was tested for patients who received CZP in P1/P2; time to loss of remission/frequency of maintenance of remission was also tested. Univariate analyses and multivariate Cox or logistic regression models were used. RESULTS: Predictors for initial remission (N = 377) included age, haematocrit, prior IBD surgery and entry HBI (P < 0.05 for all). Predictors for loss of remission (N = 437) included HBI, serum albumin concentration, haematocrit, smoking status and exposure. Predictors of maintenance of remission (N = 437) included haematocrit, IBD surgery, HBI, disease duration, serum albumin concentration and exposure. Significant predictors were confirmed with stepwise multivariate regression models. CONCLUSIONS: These analyses identified several influential parameters for short-and long-term remission of Crohn's disease with certolizumab pegol treatment. The data yield valuable hypotheses regarding factors that influence certolizumab pegol treatment. More investigation is needed. (ClinicalTrials.gov identifier NCT00552058).

Enhanced production of human recombinant proteins from CHO cells grown to high densities in macroporous microcarriers.

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (ß-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both ß-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q (P)) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q (P) with increasing cell densities up to a threshold of >1 × 10(8) cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q (P) any further. A positive linear correlation (r (2) = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 10(6) to 3 × 10(8) cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.

High productivity of human recombinant beta-interferon from a low-temperature perfusion culture.

Recombinant human interferon-beta (ß-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32°C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37°C. The percentage aggregation of ß-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.

Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions.

The enhancement of recombinant protein expression of a transfected cell line is essential for the development of an efficient large-scale bioprocess. The effect of various media additives and temperature conditions were studied in an attempt to optimize protein production, stability, and protein glycosylation from a Chinese hamster ovary (CHO) cell line producing human beta-interferon (Hu-beta-IFN). We observed a decrease in the ELISA response of the glycoprotein in the later stages of batch cultures, which was attributed to molecular aggregation. Cells were subjected to various concentrations of glycerol, dimethyl sulfoxide (DMSO), and sodium butyrate (NaBu) in a variety of culture systems and conditions. The addition of both NaBu and DMSO resulted in higher specific productivities but reduced growth rates that resulted in a net reduction of interferon produced. Glycerol appeared to stabilize the secreted beta-IFN, resulting in reduced aggregation, despite a decrease in cell growth rate. Glycosylation analysis of isolated beta-IFN showed a time-dependent decrease in sialylation in batch culture that was ameliorated by the presence of glycerol. Low-temperature conditions (30 degrees C) had the greatest effect on productivity with a significant increase in beta-IFN titer as well as a reduction in the degree of molecular aggregation.

Etiology of cortical and white matter lesions in cyclosporin-A and FK-506 neurotoxicity.

BACKGROUND AND PURPOSE: The etiology of the neurotoxicity associated with cyclosporin-A (CsA) and FK-506 treatment is not fully understood. At our institution, we noticed a distinct, abrupt change in the imaging characteristics of CsA and FK-506 neurotoxicity, which consisted of a shift in lesion morphology from a white matter abnormality to a mixed cortical and white matter pattern. The purpose of this study was to assess clinical parameters that might explain this change. METHODS: Twenty-two patients had a neurotoxic reaction and brain imaging changes while receiving CsA or FK-506. Nineteen patients received allogeneic bone marrow transplants, and three had aplastic marrow disorders. Fifty-one imaging studies (CT or MR imaging) were obtained, and lesion characteristics, locations, and time courses were evaluated along with relevant clinical data. RESULTS: Nine patients who had been conditioned for transplantation with cyclophosphamide and chemotherapy (busulfan or thiotepa) had a mixed pattern of cortical and white matter involvement (57 lesions). Isolated white matter involvement (62 lesions) developed in three nontransplant patients and 10 transplant patients conditioned with cyclophosphamide and total-body irradiation. All lesions occurred at typical brain watershed zones. Lesion enhancement was noted in two patients conditioned with chemotherapy. Initial images demonstrated characteristic lesions in 15 patients (68%). Initial images were normal in four patients (18%) and nonspecific in three patients (14%). CONCLUSION: Lesion location in CsA and FK-506 neurotoxicity may depend on the presence or type of conditioning used before bone marrow transplantation. Nontransplant patients or those conditioned with total-body irradiation develop white matter lesions, whereas those conditioned with chemotherapy develop mixed cortical and white matter lesions.

The adult radiographic shuntogram.

We describe the adult radiographic shuntogram, a simple method to evaluate the function and patency of a ventriculoperitoneal or ventriculoatrial shunt. The procedure involves placing contrast material into the valve of a shunt system and following the flow for appropriate clearing of contrast agent from the shunt tubing. Twenty-three studies were obtained in 15 patients in whom shunt malfunction was suspected. The method can be used to establish valve malfunction, ventricular or distal catheter obstruction, and peritoneal encystment.

Isolated deep cerebral venous thrombosis treated by direct endovascular thrombolysis.

BACKGROUND: Isolated thrombosis of the deep cerebral venous system is very rare and is associated with a poor prognosis. Antithrombin III (AT III) deficiency is a disorder of hypercoagulability associated with deep venous thrombosis and recurrent pulmonary emboli. We report a case of an 18-year-old man who presented with spontaneous thrombosis of the deep cerebral veins and straight dural sinus as the initial presentation of a previously undiagnosed AT III deficiency. METHODS: The patient was managed using direct endovascular infusion of the fibrinolytic agent urokinase followed by intravenous heparin. RESULTS: The technique was successful in establishing patency of the deep cerebral venous system. The patient experienced a good clinical outcome. CONCLUSIONS: Direct endovascular thrombolysis is a potentially effective management strategy for isolated thrombosis of the deep cerebral venous system.

Cost-effectiveness of initial therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors to treat hypercholesterolemia in a primary care setting of a managed-care organization.

From January 1994 through May 1995, Prudential HealthCare-North Texas prospectively studied 299 member patients diagnosed with hypercholesterolemia for whom pharmacotherapy with one of four 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, also known as statins, was prescribed. The purpose of this study was to measure the relative cost-effectiveness (CE) of these drugs in a real-world setting. This study provides information to assist decision makers in managed-care organizations (MCO) in making formulary selections. The study used a prospective, randomized, balanced cohort design, examining patients who had been prescribed initial therapy with a statin drug as monotherapy. Costs (direct medical and indirect costs) and effectiveness (percent reduction in low-density lipoprotein cholesterol levels) were based on approximately the first 6 months of initial therapy. Both the MCO and patient perspectives were considered. In the base case, mean CE ratios were significantly lower for fluvastatin compared with lovastatin, pravastatin, and simvastatin from both the managed-care perspective and the patient perspective. Sensitivity analysis did not alter the CE conclusions, even under conditions of varying cost structures. Although differences were found in the effectiveness of lovastatin, pravastatin, and simvastatin measured in this study versus efficacy measured for these drugs in controlled clinical trials, sensitivity analysis suggests that these differences alone do not determine the superior CE of fluvastatin. Finally, this study supports the idea that well-designed formularies should consider drug CE (based on safety, effectiveness, and cost) and that integration of the pharmacy benefit management with other medical management is essential. These results provide evidence that fluvastatin may represent a more cost-effective formulary choice among statin products used for initial monotherapy of hypercholesterolemia.

Endovascular thrombolysis in deep cerebral venous thrombosis.

We present two cases of acute thrombosis of the internal cerebral veins, vein of Galen, and straight sinus without sagittal sinus involvement. Both patients had hydrocephalus and severe edema of the basal ganglia and thalami, one with hemorrhagic infarction of the thalamus. Because both patients rapidly deteriorated to a comatose state, endovascular thrombolysis was performed with urokinase infusion of the deep venous structures. Thrombolysis was continued until a patent channel with brisk flow in the venous structures was achieved. Both patients survived with minimal neurologic deficits.

Paradoxical aggravation of vasospasm with papaverine infusion following aneurysmal subarachnoid hemorrhage. Case report.

Reports of intraarterial papaverine infusion as treatment for cerebral vasospasm are few and documented complications are uncommon. The authors report the case of a patient with paradoxical aggravation of cerebral arterial narrowing during selective intraarterial papaverine infusion intended to treat vasospasm following aneurysmal subarachnoid hemorrhage (SAH). A 48-year-old man presented to the authors' service with symptomatic vasospasm 10 days after experiencing an SAH. The ruptured anterior communicating artery aneurysm was surgically obliterated the following day, and thereafter maximum hypervolemic and hypertensive therapies were used. However, the patient remained lethargic, and a stable xenon-computerized tomography (CT) cerebral blood flow (CBF) study revealed CBF to be 15 cc/100 g/minute in the left anterior cerebral artery (ACA) and 25 cc/100 g/minute in the right ACA territories. Cerebral arteriography demonstrated diffuse severe left ACA and mild left middle cerebral artery (MCA) vasospasm. In response intraarterial papaverine was infused into the internal carotid artery just proximal to the ophthalmic artery. During the infusion the patient became aphasic and exhibited right hemiplegia. Arteriography performed immediately after the intraarterial papaverine infusion revealed diffuse exacerbation of vasospasm in the distal ACA and MCA territories. A repeat xenon-CT CBF study showed that CBF in the left ACA and the MCA had drastically decreased (2 cc/100 g/minute and 10 cc/100 g/minute, respectively). Despite aggressive management, infarction ultimately developed. This is the first clinical case to illustrate a paradoxical effect of intraarterial papaverine treatment for vasospasm following aneurysmal SAH. The possible mechanisms of this paradoxical response and potential therapeutic reactions are reviewed.

Angioplasty of the occluded internal carotid artery.

PURPOSE: To review patients who have presented with acute strokes from a middle cerebral artery occlusion in whom in addition to the middle cerebral artery thromboembolus, an internal carotid artery occlusion has been present, and in whom angioplasty of these totally occluded internal carotid arteries has bee n successful. METHODS: We reviewed retrospectively our experience in treating a cute stroke patients with intracranial, intraarterial urokinase. Six of 27 patients had internal carotid artery occlusions in addition to middle cerebral artery occlusions. Two patients presented with spontaneous carotid dissections for wh ich no further intervention from the ipsilateral internal carotid artery was attempted. In the remaining four internal carotid artery occlusions secondary to atherosclerotic disease, standard guide wires and catheters were negotiated across the level of the internal carotid artery occlusion, which expedited intracranial catheterization for thrombolysis. Subsequently, angioplasty of the internal carotid artery was performed. RESULTS: All four occluded internal carotid arteries could be traversed. No new neurologic deficits occurred. No vascular injuries occurred. No deaths occurred. Four- to 6-month follow-up showed all four internal carotid arteries remained patent. CONCLUSION: In acute occlusions of the internal carotid artery from atherosclerosis, the occluded vessel can sometimes be recanalized with low morbidity. In addition, endovascular access to the intracranial circulation can be expedited by using the recanalized internal carotid artery.

Computed tomography-directed fine needle aspiration of skull base parapharyngeal and infratemporal fossa masses.

Suspicious findings in the parapharyngeal region on computed tomographic (CT) or magnetic resonance imaging studies can be a diagnostic problem. Blind biopsy through the mucosa can be inadequate, since the abnormality is not directly visible. With CT guidance, fine needle aspiration (FNA) of parapharyngeal masses can be performed with a needle confidently placed within the lesion. Vital structures such as the carotid artery are avoided. We present a series of 33 CT-guided FNA on 30 patients to evaluate the safety and the degree of accuracy of the procedure. Most of the patients had been treated previously for local malignancy. All patients had surgical pathologic study, autopsy, or clinical and imaging follow-up to confirm the FNA cytology results. Twenty of the 33 biopsies were positive for malignant cells, confirming recurrence of the primary head and neck malignancy. Of the 33 CT-directed FNA, 13 were negative for malignant cells. Three of these 13 were found to be false-negative FNA. None of the patients had complications from the procedure. CT directed FNA of masses at the skull base or in the parapharyngeal area can be performed safely. A high degree of accuracy is achieved, with 30 (90.9%) accurate in identifying the presence or absence of malignancy in our series.

Antisense oligodeoxyribonucleotide inhibition of TGF-beta 1 gene expression and alterations in the growth and malignant properties of mouse fibrosarcoma cells.

Transforming growth factor (TGF-beta) is a family of multifunctional signalling molecules that play a fundamental role in both normal and malignant cell behavior. Procedures that alter mouse TGF-beta 1 gene expression provide an important approach for analyzing the complex regulatory processes associated with this member of the growth factor family. Therefore, we have designed oligodeoxyribonucleotides (oligos) in an antisense orientation, which are complementary to regions of the TGF-beta 1 message, in an attempt to obtain an oligo sequence that specifically reduces TGF-beta 1 synthesis. We observed that oligos containing a mixture of phosphorothioate and phosphodiester linkages were less toxic and more specific when compared to those only containing phosphorothioate. A non-toxic sequence was identified that markedly reduced the levels of TGF-beta 1 in oligo-treated malignant mouse fibrosarcoma cells. The invasive and metastatic properties of these fibrosarcoma cells were also significantly decreased following treatment with the antisense oligo. The results indicate an important role for altered TGF-beta 1 expression in the regulation of malignant cell proliferation, invasion and metastasis. These results also indicate that this oligo sequence is a useful tool for studies directed towards understanding the complex relationships between TGF-beta 1 and cellular regulation.

Is bilateral lower extremity compression sonography useful and cost-effective in the evaluation of suspected pulmonary embolism?

OBJECTIVE: Because deep venous thrombosis is clinically linked with pulmonary embolism and often treated similarly, we sought to assess the usefulness of obtaining bilateral lower extremity compression sonograms when findings on ventilation-perfusion lung scans indicate a low or indeterminate probability of pulmonary embolism. Demonstration of deep venous thrombosis would provide a rationale for treating both pulmonary embolism and deep venous thrombosis. MATERIALS AND METHODS: Two hundred twenty-three consecutive patients with suspected pulmonary embolism had ventilation-perfusion lung scans and concurrent bilateral lower extremity compression sonograms; 34 also had pulmonary arteriography. RESULTS: In 75 cases, the results of ventilation-perfusion lung scanning indicated an indeterminate probability of pulmonary embolism. Evidence of thrombosis was seen on sonograms in 11 of these 75. In the remaining 64, 17 underwent pulmonary arteriography and four (24%) had pulmonary embolism. Findings on lung scans indicated a low probability of pulmonary embolism in 70 of 223 patients. Evidence of thrombosis was seen on sonograms in 11 of these 70. Five of the remaining 59 underwent pulmonary arteriography and one (20%) had pulmonary embolism. According to the 1993 Medicare Fee Schedule, if all 145 patients whose lung scans were nondiagnostic had sonography and only those with normal sonograms had pulmonary arteriography, the professional and hospital charges would be $359,552. If all 145 had pulmonary arteriography without sonography, the charges would be $395,031. CONCLUSION: If ventilation-perfusion lung scans indicate a low or an indeterminate probability of pulmonary embolism and bilateral lower extremity compression sonography is performed, only those patients with normal sonographic findings would need further study. Thus, 15% (22/145) of patients could be spared pulmonary arteriography, and the estimated savings in cost would be 9%.

TGF-beta 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan.

TGF-beta is a potent stimulator of motility in a variety of cell types. It has recently been shown that hyaluronan (HA) can directly promote locomotion of cells through interaction with the HA receptor RHAMM. We have investigated the role of RHAMM and HA in TGF-beta-stimulated locomotion and show that TGF-beta triggers the transcription, synthesis and membrane expression of the RHAMM receptor and the secretion of HA coincident with the induction of the locomotory response. This was demonstrated by both incubating cells with exogenous TGF-beta 1 and by stimulating the production of bioactive TGF-beta 1 in tumor cells transfected with TGF-beta 1 under the control of the metallothionein promoter. TGF-beta 1-induced locomotion was suppressed by antibodies that prevented HA/RHAMM interaction, using polyclonal antibodies to either RHAMM fusion protein or RHAMM peptides, or mAbs to purified RHAMM. Peptides corresponding to the HA-binding motif of RHAMM also suppressed TGF-beta 1-induced increases in motility rate. Spontaneous locomotion of fibrosarcoma cells was blocked by neutralizing secreted TGF-beta with panspecific TGF-beta antibodies and by inhibition of TGF-beta 1 secretion with antisense oligonucleotides. Polyclonal anti-RHAMM fusion protein antibodies and peptide from the RHAMM HA-binding motif also suppressed the spontaneous motility rate of fibrosarcoma cells. These data suggest that fibrosarcoma cell locomotion requires TGF-beta, and the pathway by which TGF-beta stimulates locomotion uses the HA receptor RHAMM and HA.

Degradation of type IV collagen during the development of fetal rat lung.

Lung structure undergoes rapid remodeling during late gestation as a functional respiratory unit is formed. To determine the role of collagen turnover in this process, particularly the basement membrane component, we studied the degradation of collagen in a series of fetal rats from day 18 of gestation to full term. During the period of rapid cell proliferation to day 20, the collagen level per milligram of lung did not change though the rate of synthesis increased. More than 40% of new collagen was rapidly degraded. At the end of the growth phase, collagen synthesis rose rapidly as the total collagen content increased in the lung. Over this period, little Type I collagenase activity could be detected, but degradation of Type IV collagen was readily measured and was maximal at 18 to 20 days. The enzyme(s) was almost all present in the active form, and evidence for dissolution of the subepithelial basement membrane was also found by electron microscopy. Using isolated fetal epithelial cells and fibroblasts, supernatants of both cell types showed degradative activity for Type IV collagen, particularly at days 18 to 20. The major enzyme involved appears to be a 72 kD collagenase, as shown by zymography and by mRNA expression in both cell types. The results demonstrate that rapid degradation of Type IV collagen occurs during the growth phase of late fetal lung development, and that both epithelial and stromal cells contribute to collagenolytic activity.

The importance of N-linked glycoproteins and dolichyl phosphate synthesis for fusion of L6 myoblasts.

Myoblasts fuse to form multinucleated myotubes, one of the early steps in the formation of multinucleated muscle fiber. The fusion reaction is accompanied by biochemical differentiation resulting in the expression of a variety of enzyme activities and macromolecules, particularly creatine phosphokinase. The fusing myoblast is thus an excellent system for use in studies on the molecular basis of cellular recognition. This report focuses on the role played by glycoproteins in this process. It was found that alteration of cell-surface glycoproteins, using oligosaccharide-processing inhibitors that interfered with the synthesis of the high-mannose type of N-linked oligosaccharide, resulted in the inhibition of both the fusion reaction and biochemical differentiation as determined by measurement of creatine phosphokinase. Ketoconazole, compactin, and lovastatin, which affect dolichol and cholesterol biosynthesis, were also potent fusion inhibitors. These observations, coupled with earlier studies on the characterization of fusion-defective myoblast cell lines defective in glycoprotein biosynthesis, point to the importance of surface glycoproteins in cellular recognition in L6 myoblasts.

The inhibition of platelet aggregation of metastatic H-ras-transformed 10T1/2 fibroblasts with castanospermine, an N-linked glycoprotein processing inhibitor.

A series of T24-H-ras-transformed 10T1/2 fibroblasts with varying metastatic potential was tested for the ability to aggregate platelets. Results indicate that although platelet activation was always detected in the highly metastatic cells, some non-metastatic cells also have the ability to cause platelet aggregation, suggesting that this is a necessary but not sufficient characteristic of the metastatic phenotype. Apyrase, an ADP scavenger, effectively inhibited platelet aggregation by metastatic cells, however, there was no significant increase in ADP secretion or relation to the ability of the tumor cells to activate platelets. Hirudin, a thrombin inhibitor, did not affect aggregation, suggesting that the pathway of activation is thrombin-independent. The glycoprotein processing inhibitor, castanospermine, which reduces glycosidase I activity and metastatic capability, inhibited the ability of metastatic cells to cause platelet aggregation. However, another inhibitor of oligosaccharide processing, swainsonine, which inhibits mannosidase II activity and does not reduce metastasis, had no effect on platelet aggregation. These results show that the integrity of N-linked oligosaccharide structure of glycoproteins is an important feature of the ability of ras-transformed fibroblasts to activate platelets.

Metabolism of azoxy derivatives of procarbazine by aldehyde dehydrogenase and xanthine oxidase.

Procarbazine, a 1,2-disubstituted hydrazine, is employed therapeutically in the treatment of Hodgkin's disease and a limited number of other neoplasias. The isomeric azoxy metabolites of procarbazine have recently been identified as the precursors of species responsible for both the anti-cancer efficacy and toxic effects mediated by this drug. This study demonstrates that cytosolic enzymes are involved in the metabolism of the azoxy metabolites of procarbazine. Two azoxy procarbazine oxidase activities were resolved by diethylaminoethyl (DEAE)-cellulose chromatography. The activity which did not bind to this column was purified to homogeneity and was identified as a phenobarbital-inducible form of cytosolic aldehyde dehydrogenase. This protein fraction was shown to metabolize only the azoxy 2 procarbazine isomer to yield N-isopropy-p-formylbenzamide (ALD) in a reaction which did not require NAD+ as cofactor. The ALD product formed was also a substrate for a subsequent NAD(+)-dependent reduction reaction catalyzed by that purified protein. The azoxy 2 procarbazine isomer and ALD were shown to be potent inhibitors of both the dehydrogenase and esterase activities of aldehyde dehydrogenase. The second azoxy procarbazine oxidase activity which was retained by the DEAE-cellulose column co-eluted with xanthine oxidase activity. Both the xanthine dehydrogenase/oxidase and azoxy procarbazine oxidase activities of this protein fraction were inhibited by allopurinol, a specific inhibitor of xanthine dehydrogenase. Xanthine dehydrogenase/oxidase was partially purified by an alternative procedure and was shown to metabolize both the azoxy 2 procarbazine isomer and ALD, ultimately producing N-isopropylterephthalamic acid. The ability of xanthine oxidase to metabolize azoxy 2 procarbazine and ALD was confirmed using commercial, purified milk xanthine oxidase.