ROC study and SUV threshold using quantitative multi-modal SPECT for bone imaging.

BACKGROUND: We investigated the clinical performance of a quantitative multi-modal SPECT/CT reconstruction platform for yielding radioactivity concentrations of bone imaging with 99mTc-methylene diphosphonate (MDP) or 99mTc-dicarboxypropane diphosphonate (DPD). The novel reconstruction incorporates CT-derived tissue information while preserving the delineation of tissue boundaries. We assessed image-based reader concordance and confidence, and determined lesion classification and SUV thresholds from ROC analysis. METHODS: Seventy-two cancer patients were scanned at three US and two German clinical sites, each contributing two experienced board-certified nuclear medicine physicians as readers. We compared four variants of the reconstructed data resulting from the Flash3D (F3D) and the xSPECT Bone™ (xB) iterative reconstruction methods and presented images to the readers with and without a fused CT, resulting in four combinations. We used an all-or-none approach for inclusion, compiling results only when a reader completed all reads in a subset. After the final read, we conducted a "surrogate truth" reading, presenting all data to each reader. For any remaining discordant lesions, we conducted a consensus read. We next undertook ROC analysis to determine SUV thresholds for differentiating benign and lesional uptake. RESULTS: On a five-point rating scale of image quality, xB was deemed better by almost two points in resolution and one point better in overall acceptance compared to F3D. The absolute agreement of the rendered decision between the nine readers was significantly higher with CT information either inside the reconstruction (xB, xBCT) or simply through image fusion (F3DCT): 0.70 (xBCT), 0.67 (F3DCT), 0.64 (xB), and 0.46 (F3D). The confidence level to characterize the lesion was significantly higher (3.03x w/o CT, 1.32x w/CT) for xB than for F3D. There was high correlation between xB and F3D scores for lesion detection and classification, but lesion detection confidence was 41% higher w/o CT, and 21% higher w/CT for xB compared to F3D. Without CT, xB had 6.6% higher sensitivity, 7.1% higher specificity, and 6.9% greater AUC compared to F3D, and similarly with CT-fusion. The overall SUV-criterion (SUVc) of xB (12) exceeded that for xSPECT Quant™ (xQ; 9), an approach not using the tissue delineation of xB. SUV critical numbers depended on lesion volume and location. For non-joint lesions > 6 ml, the AUC for xQ and xB was 94%, with SUVc > 9.28 (xQ) or > 9.68 (xB); for non-joint lesions ≤ 6 ml, AUCs were 81% (xQ) and 88% (xB), and SUVc > 8.2 (xQ) or > 9.1 (xB). For joint lesions, the AUC was 80% (xQ) and 83% (xB), with SUVc > 8.61 (xQ) or > 13.4 (xB). CONCLUSION: The incorporation of high-resolution CT-based tissue delineation in SPECT reconstruction (xSPECT Bone) provides better resolution and detects smaller lesions (6 ml), and the CT component facilitates lesion characterization. Our approach increases confidence, concordance, and accuracy for readers with a wide range of experience. The xB method retained high reading accuracy, despite the unfamiliar image presentation, having greatest impact for smaller lesions, and better localization of foci relative to bone anatomy. The quantitative assessment yielded an SUV-threshold for sensitively distinguishing benign and malignant lesions. Ongoing efforts shall establish clinically usable protocols and SUV thresholds for decision-making based on quantitative SPECT.

Metal-activated C-peptide facilitates glucose clearance and the release of a nitric oxide stimulus via the GLUT1 transporter.

AIMS/HYPOTHESIS: Proinsulin C-peptide has been implicated in reducing complications associated with diabetes and also in improving blood flow. We hypothesised that incubation of erythrocytes with C-peptide would improve the ability of these cells to release ATP, a stimulus for nitric oxide production. METHODS: Erythrocytes obtained from rabbits (n = 11) and both healthy and type 2 diabetic humans (n = 7) were incubated with C-peptide in the absence and presence of Fe2+ and Cr3+, and the resulting ATP release was measured via chemiluminescence. This release was also measured in the presence and absence of phloretin, an inhibitor of GLUT1, and also of mannose, a glycolysis inhibitor. To determine glucose transport, 14C-labelled glucose was added to erythrocytes in the presence and absence of the C-peptide-metal complex and the aforementioned inhibitors. RESULTS: The release of ATP from the erythrocytes of patients with diabetes increased from 64 +/- 13 to 260 +/- 39 nmol/l upon incubation of the cells in C-peptide. The C-peptide activity was dependent upon binding to Fe2+, which was extended upon binding to Cr3+. The increase in ATP release from the erythrocytes is due to metal-activated C-peptide stimulation of glucose transfer into the erythrocytes via the GLUT1 transporter. In the presence of C-peptide complexed to Cr3+, the amount of glucose transferred into the erythrocyte increased by 31%. CONCLUSIONS/INTERPRETATION: When complexed to Fe2+ or Cr3+, C-peptide has the ability to promote ATP release from erythrocytes. This release is due to an increase in glucose transport through GLUT1.

[Long-term results after balloon angioplasty of the crural artery]. / Langzeitergebnisse nach Ballonangioplastie kruraler Arterien.

PURPOSE: To evaluate the technical success rate, procedure-related complications, and clinical long-term results for patients who underwent infrapopliteal angioplasty. MATERIALS AND METHODS: We retrospectively evaluated all patients who underwent infrapopliteal angioplasty to treat critical chronic limb ischemia or severe claudication from 1/1997 to 12/1999. We excluded patients with acute (< 2 weeks) limb ischemia. Procedure-related data were prospectively documented in a database and analyzed with a focus on the technical success rate and procedure-related complications. In addition all clinical documents were analyzed, and a follow-up examination was performed or telephone interviews were conducted with patients, relatives and referring doctors for follow-up. The primary end points were the limb salvage rate and patient survival rate. The secondary end points included the complication rate, technical success rate, and walking distance. RESULTS: 112 patients with a mean age of 72 years (41 women, 71 men) underwent crural angioplasty on 121 limbs. Four patients suffered from severe claudication (Rutherford category 3) and all others had critical chronic limb ischemia (category 4 to 6). The complication rate was 2.7 %. The technical success rate was 92 %. The ankle brachial index increased from 0.59 to 0.88. The mean walking distance increased significantly from 52 +/- 66 to 284 +/- 346 meters at the time of follow-up. The limb salvage rate was 83.6 % after one year and 81.1 % after three years. The mean survival rate according to Kaplan-Meier was 79.4 %, 69.2 %, and 54.2 % at 1, 2, and 3 years, respectively. Patients with at least one patent run-off vessel after angioplasty had a significantly better limb salvage rate. Diabetes was not a risk factor for limb salvage. CONCLUSION: Infrapopliteal angioplasty shows a high technical success rate with an acceptable complication rate. The clinical long-term success seems favorable if a least one open run-off vessel was able to be achieved. However, the survival rate was limited by the co-morbidity in this patient group.

[Treatment of malignant biliary occlusion by means of transhepatic percutaneous biliary drainage with insertion of metal stents--results of an 8-year follow-up and analysis of the prognostic parameters]. / Behandlung der malignen Gallenwegsstenose mittels perkutaner transhepatischer Metallendoprothesenimplantation: 8 Jahres-Ergebnisse und Analyse prognostischer Faktoren.

PURPOSE: To analyze outcome and predictive factors for patient survival and patency rates of unresectable malignant biliary obstruction treated with percutaneous transhepatic insertion of metal stents. MATERIALS AND METHODS: This is a retroslective analysis of 130 patients treated in one interventional radiological center with data collected from patient records and by telephone interviews. The procedure-related data had been prospectively documented in a computer data base. The Kaplan-Meier analysis was performed for univariate and multivariate comparison of survival and patency rates with the log-rank test used for different tumor types. Predictive factors for survival and 30-day mortality were analyzed by a stepwise logistic regression. RESULTS: Underlying causes of malignant biliary obstructions were cholangiocarcinoma in 50, pancreatic carcinoma in 29, liver metastases in 27, gallbladder carcinoma in 20, and other tumors in 4 patients. The technical success rate was 99 %, the complication rate 27 % and the 30-day mortality 11 %. Primary patency rates (406 days with a median of 207 days) did not differ significantly for different tumor types. The survival rates were significantly (p = 0.03 by log-rank test) better for patients with cholangiocarcinoma than for patients with pancreatic carcinoma and liver metastases. Multiple regression analysis revealed no predictive factor for patient survival and 30-day mortality. CONCLUSION: Percutaneous transhepatic insertion of metal biliary endoprostheses offers a good initial and long-term relief of jaundice caused by malignant biliary obstruction. Although survival rates for patients with cholangiocarcinoma are better than for other causes of malignant biliary obstruction, a clear predictive factor is lacking for patients undergoing palliative biliary stent insertion.

Gelastic fits in a two years old boy with a complex cerebral malformation.

We report the case of a two years old boy showing gelastic fits as the leading clinical symptom of a rare complex cerebral malformation with closed-lip schizencephaly, an arachnoid cyst and a partial agenesis of the corpus callosum. After 5 uneventful interictal electroencephalograms the patient underwent 24 h EEG with telemetry while finally presenting two laughing fits with associated spike-wave discharges in the right-centroparietal region. Medical treatment with carbamacepine was initiated and the seizure frequency decreased significantly. This is the first cited case of gelastic fits occurring in schizencephaly. In contrast to previously cited cases with laughing fits we observed a good response to anticonvulsive treatment. Moreover our patient presented a surprisingly favourable psycho-mental and motor development during an 8 months observation period.

Multiplanar reconstructions and three-dimensional imaging (computed rotational osteography) of complex fractures by using a C-arm system: initial results.

With use of a calibrated angiographic C-arm system and a postprocessing workstation, the authors acquired volume data sets from two-dimensional digital projection images obtained during a C-arm rotation around the patient axis. Multiplanar reconstruction and three-dimensional images of complex fractures were reconstructed and compared with spiral computed tomographic studies in a cadaveric pig study and in eight patients. Computed rotational osteography provided high-resolution multiplanar reconstruction and three-dimensional images of complex fractures.

CT-fluoroscopy: Tool or gimmick?

Recent advances in CT scanner technology and computer hardware have led to the development of CT fluoroscopy (CTF), which allows real-time acquisition and display of cross-sectional images (with a rate of up to 8 frames per second). Since the introduction of the first CT fluoroscopy scanner in 1993, a variety of these scanners have been installed world-wide and many reports on the clinical use of this device have appeared recently. However, use of this new technology for the guidance of interventional radiologic procedures, such as percutaneous biopsy and percutaneous drainage, is not uniformly advocated by interventional radiologists. Concerns have been reported regarding radiation exposure and outcome of the procedures when compared with sequential CT guidance or other alternative guiding modalities. This article is intended to present an overview of CTF technology, to summarize the results of published papers on various interventional applications and to reflect on its specific advantages and disadvantages.

Engineering of human vascular aortic tissue based on a xenogeneic starter matrix.

BACKGROUND: The goal for tissue engineering of vascular grafts is the replacement of a diseased vessel with a functional and stable graft. We now introduce a new concept for the tissue engineering of vessels. The idea was to humanize a previously acellularized, but structurally intact, xenogeneic vessel by repopulation with human autologous cells. To this purpose, a gentle nondenaturing and nondeterging acellularization procedure for xenogeneic aortas was developed. This structure was reseeded with pre-expanded peripheral vascular endothelial cells (EC) and myofibroblasts using specifically designed bioreactors. METHODS: Aortas from 15-30 kg female landrace pigs were acelullarized with a 0.1% trypsin solution for between 24 and 96 hr. Human vascular cells were harvested from saphenous vein biopsy specimens. Acellularized vessels were reseeded with EC and myofibroblasts. Cell viability after reseeding was assayed by fluorescence staining. Morphologic features of the acellularized matrix and tissue engineered vessel was assayed by transmission and scanning electron microscopy and histologic analysis. Nitric oxide-synthetase activity was investigated by mass spectrometric analysis of bioreactor supernatants. The in vivo immune response was tested by subcutaneous implantation of acellularized porcine aortic tissue in a rat model. RESULTS: The acellularization procedure resulted in an almost complete removal of the original resident cells, and the 3-D matrix was loosened at interfibrillar zones. However, the 3-D arrangement of the matrix fibers was grossly maintained. The 3-D matrix was covered with a fully confluent human endothelial cell layer obtained by continuous stress challenge in the bioreactor. Myofibroblasts migrated into positions formerly occupied by the xenogeneic cells. Nitric oxide synthetase activity was maintained in the bioartificial graft. T-lymphocyte and CD18 positive leukocyte infiltrate were greatly reduced after acellularization of porcine aortic specimens after implantation in the rat. CONCLUSIONS: Porcine vessels were acellularized and consecutively fully repopulated with human EC and myofibroblasts. This approach may eventually lead to the engineering of vessels immunologically acceptable to the host using a relatively short preparation period of 2-3 weeks. We expect matrix turnover in vivo leading to a gradual assimilation of the matrix structure by the host mediated by the hosts autologous cells.

A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus.

BACKGROUND: Chloroplast division in plant cells occurs by binary fission, yielding two daughter plastids of equal size. Previously, we reported that two Arabidopsis homologues of FtsZ, a bacterial protein that forms a cytokinetic ring during cell division, are essential for plastid division in plants, and may be involved in the formation of plastid-dividing rings on both the stromal and cytosolic surfaces of the chloroplast envelope membranes. In bacteria, positioning of the FtsZ ring at the center of the cell is mediated in part by the protein MinD. Here, we identified AtMinD1, an Arabidopsis homologue of MinD, and investigated whether positioning of the plastid-division apparatus at the plastid midpoint might involve a mechanism similar to that in bacteria. RESULTS: Sequence analysis and in vitro chloroplast import experiments indicated that AtMinD1 contains a transit peptide that targets it to the chloroplast. Transgenic Arabidopsis plants with reduced AtMinD1 expression exhibited variability in chloroplast size and number and asymmetrically constricted chloroplasts, strongly suggesting that the plastid-division machinery is misplaced. Overexpression of AtMinD1 inhibited chloroplast division. These phenotypes resemble those of bacterial mutants with altered minD expression. CONCLUSIONS: Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD. The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.

Comparison of C-arm CT fluoroscopy and conventional fluoroscopy for percutaneous biliary drainage procedures.

PURPOSE: To conduct a prospective randomized evaluation of C-arm computed tomography (CT) fluoroscopy for external biliary drainage procedures in comparison with conventional fluoroscopic guidance to reduce the number of transhepatic punctures as a primary endpoint. MATERIALS AND METHODS: In 18 patients with biliary obstructions, 20 external percutaneous biliary drainage procedures were prospectively performed with use of either C-arm CT fluoroscopy or conventional fluoroscopy alone. The number of hepatic punctures, procedure time, and fluoroscopy time, were analyzed separately for both methods. RESULTS: C-arm CT fluoroscopy resulted in a reduced number of transhepatic punctures, with decreased procedure and fluoroscopy times (P < .05; t test). When compared with conventional external biliary drainage procedures, a mean of 1.8+/-1 versus 4.8+/-2.8 hepatic punctures at a fluoroscopy time of 3.4+/-1.5 versus 11.4+/-7.4 minutes was required for C-arm CT fluoroscopy, while procedure times were 11+/-3.6 versus 16.2+/-9.3 minutes. CONCLUSIONS: C-arm CT fluoroscopy is associated with decreased procedure and fluoroscopy times, while fewer transhepatic punctures are required to establish external biliary drainage.

Feasibility of C-arm-supported CT fluoroscopy in percutaneous abscess drainage procedures.

PURPOSE: Evaluation of C-arm-supported CT fluoroscopy to facilitate percutaneous abscess drainage procedures. METHODS: Prospectively, 40 percutaneous drainage procedures were performed either with C-arm-supported CT fluoroscopy or with CT fluoroscopy alone. Hybrid imaging was performed on the CT couch after complementing a CT fluoroscopy scanner with a C-arm fluoroscopy unit. Procedure times, drainage revisions during follow-up, and postinterventional drainage periods were analyzed. RESULTS: When compared with exclusive CT fluoroscopic guidance, a median procedure time of 9 +/- 3.7 min versus 14.8 +/- 7.3 min was required for C-arm-supported CT fluoroscopy (p < 0.005, t-test). During follow-up, eight drainage catheters had to be revised within the exclusive CT fluoroscopy group, while only two revisions were necessary within the C-arm-supported CT fluoroscopy group. With C-arm-supported CT fluoroscopy, postinterventional drainage periods were reduced (median 13 vs 19 days; p < 0.001, t-test). CONCLUSION: Compared with exclusive cross-sectional image guidance, C-arm-supported CT fluoroscopy seems to improve placement of abscess drainage catheters to possibly reduce procedure times, drainage catheter revisions, and postinterventional drainage periods.

Effects of acute euglycemic hyperinsulinemia on urinary nitrite/nitrate excretion and plasma endothelin-1 levels in men with essential hypertension and normotensive controls.

Insulin stimulates the production of endothelin-1 (ET-1) and nitric oxide (NO) by isolated endothelial cells. Additionally, insulin-dependent glucose transport and insulin-mediated NO production partially share common elements in signal transduction. There are discordant data on plasma ET-1 levels during acute euglycemic systemic hyperinsulinemia in normotensive men and men with essential hypertension (EH) (known to be insulin-resistant), as well as on the relations between insulin sensitivity and vascular function. Our aim was to assess the response of approximate measures of whole-body generation of NO and ET-1 to acute euglycemic hyperinsulinemia in EH patients and controls. We studied 17 newly diagnosed untreated men with uncomplicated EH and 10 normotensive controls. Plasma ET-1 and urinary excretion of nitrite plus nitrate, stable NO metabolites (Uno(x)), were measured before and during a 3-hour hyperinsulinemic-euglycemic clamp. Both in hypertensives and normotensives, plasma ET-1 levels were reduced after 2 hours of the clamp (EH: baseline, 3.1+/-1.9 pg/mL; 2 hours, 1.9+/-1.2 pg/mL, P = .04 v baseline; controls: baseline, 4.2+/-2.6 pg/mL; 2 hours, 2.8+/-1.4 pg/mL, P = .04 v baseline). No significant changes in Uno(x) during the clamp were observed. Changes in Uno(x) during the clamp (deltaUno(x)) and differences in plasma ET-1 measured before the end and before the beginning of the clamp (deltaET-1) were correlated in the controls (r = .75, P = .01) but not in EH (r = -.01, P = .97). No parameter of glucose metabolism correlated with basal Uno(x), basal plasma ET-1, deltaUno(x), and deltaET-1, whether absolute or percent values, in either group. Thus, acute euglycemic hyperinsulinemia produces a decrease in plasma ET-1 in both EH patients and controls. The lack of correlation between deltaUno(x) and deltaET-1 under these conditions in EH may suggest an impairment of systems governing interactions between the NO-dependent pathway and ET-1. In addition, insulin actions on glucose metabolism and on the endothelial mediators appear dissociated.

The influence of radiation and chemotherapy-related DNA strand breaks on carcinogenesis: an evaluation.

PURPOSE: DNA strand breaks are believed to induce carcinogenesis. This study was conducted to analyze induction and repair of irradiation- and chemotherapy-related strand breaks in vitro. METHODS: Friend Leukemia cells were exposed to irradiation and various chemotherapeutic agents at different doses and concentrations. Occurrence of strand breaks was determined fluorometrically, measuring the rate of DNA unwinding immediately after exposure and 24 hours later. RESULTS: The amount of double-stranded DNA decreased significantly for irradiation, doxorubicin, dactinomycin and etoposide (p < or = 0.05, t-test). After 24 hours free of exposure, the persistent damage was detectable for all of these agents but not for irradiated cells, with DNA strand breaks being decreased for etoposide, unchanged for doxorubicin and increased for methotrexate as well as for dactinomycin. CONCLUSIONS: Severe DNA damage is induced by various chemotherapeutic agents and by irradiation. While repair of chemotherapy-related strand breaks may remain incomplete or prolonged for some chemotherapeutic agents, repair of radiation induced strand breaks is faster and more complete. Therefore chemotherapy-related carcinogenesis may partially be explained by prolonged persistence of DNA strand breaks.

[Pulmonary, nodular cavernous, calcifying form of amyloidosis]. / Pulmonale, noduläreinschmelzende, kalzifizierende Form der Amyloidose.

We report a rare case of pulmonary nodular amyloidosis featuring all typical morphologic alterations associated with this disease; multiple amyloidomas, calcification and cavernous transformation were present. Definite diagnosis was established histologically with CT-guided biopsy. The different types of pulmonary amyloidosis and their radiological appearances are described and discussed.

Effects of nitinol Strecker stent placement on vascular response in normal and stenotic porcine iliac arteries.

PURPOSE: This experimental study was conducted to evaluate neointimal thickness, lumen diameters, and histologic changes in normal and stenotic porcine iliac arteries following placement of self-expanding nitinol Strecker stents. MATERIALS AND METHODS: Neointimal trauma causing slight vascular stenosis was induced unilaterally within external iliac arteries of 12 swines by means of endothelial abrasion and high cholesterol diet. Nitinol Strecker stents were placed within the stenotic and the normal contralateral vascular segments. For histopathologic evaluation, the pigs were killed 12 or 24 weeks after stent placement and luminal diamters were evaluated angiographically. RESULTS: Excluding one occlusion, 15% narrowing of the lumen diameter was induced unilaterally (P = .002). Initial luminal gain after stent placement was greater for stenotic than for normal arteries. The amount of neointima thickness was not different between stenotic and normal vessels (P > .05). Comparing vascular diameters before stent placement and at follow-up, luminal loss due to neointima proliferation was 22% within normal arteries (P = .0002), while a luminal gain by 15% was found within the stenotic arteries (P = .008). Maturation of neointima and endothelial coverage were complete after 24 weeks. CONCLUSIONS: Even though nitinol Strecker stents induce excessive neointimal proliferation, stenotic arteries seem to profit from great early luminal gain resulting in 15% of vascular expansion at follow-up while slight stenosis is induced within normal iliac arteries.

[Control of percutaneous biopsy with CT fluoroscopy]. / Steuerung von perkutanen Biopsien mittels CT-Fluoroskopie.

PURPOSE: Clinical evaluation of CT fluoroscopy and comparison with conventional CT guidance for monitoring of non-pulmonary percutaneous biopsy procedures. MATERIALS AND METHODS: 20 non-pulmonary CT-guided biopsy procedures were prospectively performed either with CT fluoroscopy or with conventional CT guidance. CT fluoroscopy was performed using 120 kV and 50, 70 or 90 mA at a frame-rate of three or six images per second. Number of punctures and biopsies, procedure times, radiation doses and histologic results were analyzed separately for conventional CT guidance and for CT fluoroscopy. RESULTS: With CT fluoroscopy, yield of biopsies was improved (p = 0.005, t-test) and procedure times were shorter than for conventional CT guidance (11.4 +/- 6.0 vs. 23.6 +/- 13.8 min; p = 0.03, t-test). Analysis of procedure related radiation exposure and histologic outcome showed no significant difference between conventional and fluoroscopic CT-guided procedures (p > 0.05, t-test). CONCLUSIONS: CT fluoroscopy facilitates guidance of percutaneous biopsy procedures. Compared to conventional CT assistance, procedure times are decreased while yield of biopsies is improved.

Reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetric dimethylarginine in men with essential hypertension.

Our aim was to investigate systemic nitric oxide (NO) production and its potential determinants such as insulin resistance, dyslipidemia, and circulating methylated analogs of L-arginine in uncomplicated essential hypertension (EH). Nineteen newly diagnosed, untreated male subjects with mild pure uncomplicated EH and 11 normotensive controls were studied at rest after an overnight fast. The groups had comparable age, body mass index, creatinine clearance, cholesterol, fasting glucose, and insulin. In hypertensives, the urinary excretion rate of nitrite plus nitrate (Unox), an index of endogenous NO production, was depressed (56+/-17 vs. 77+/-23 micromol/mmol creatinine; p < 0.05), whereas plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, were increased (2.4+/-1.1 vs. 1.1+/-0.7 microM; p < 0.005). Circulating concentrations of symmetric dimethylarginine were similar in both groups (1.4+/-1.3 vs. 1.5+/-1.1 microM; p = NS). The L-arginine-to-ADMA ratio was reduced in hypertension (3.3+/-0.5 vs. 4.5+/-0.8; p < 0.001 for In-transformed data). There was no correlation between Unox and either the magnitude of insulin resistance or dyslipidemia in EH. Thus in male subjects with EH, endogenous systemic NO formation appears depressed, which is unrelated to accompanying insulin resistance or dyslipidemia. Circulating ADMA levels are increased in uncomplicated EH, which may be of potential relevance.

Chitobiase, a new reporter enzyme.

N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-beta-D-glucosaminidase activities are missing in E. coli strains, (ii) chitobiase can be monitored using blue/white colony indicator plates and (iii) convenient substrates for this enzyme are commercially available. The use of chitobiase as a reporter enzyme is generally applicable to the study of gene expression in those bacteria that do not contain N-acetyl-beta-D-glucosaminidases. We constructed plasmid vectors containing a multiple cloning site for producing in-frame fusions to chitobiase, the attP of lambda phase for movement into the bacterial chromosome for single-copy analysis, the gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 origin of replication and the rrnBt1t2 terminator region upstream of the chb gene to prevent read-through from other promoters. In-frame fusions between the dnaA gene and chb were moved to the chromosome by site-specific recombination with the chromosomal attB site. These single-copy fusions were assayed for chitobiase to examine the effects of a deletion in the dnaA regulatory region.