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At least 127 records · Page 7Linked to original sources

Cell cycle phase-specific cDNA libraries reflecting phase-specific gene expression of Ehrlich ascites cells growing in vivo.

Asynchronous populations of Ehrlich ascites tumor cells grown in vivo were separated by centrifugal elutriation into fractions of G1-, S-, and G2/M-phase cells with less than 10% cross-contamination. Cytoplasmic mRNA from phase-synchronous cells was used to prepare cDNA which was ligated with bacteriophage lambda gt10 arms and amplified in Escherichia coli C600 hfl-. EcoRI digests of DNA isolated from the sublibraries (G1, S, G2/M) were submitted to Southern hybridizations with radiolabeled probes either (a) for genes whose phase-specific expression is clearly documented, thymidine kinase, dihydrofolate reductase, and thymidylate synthase, or (b) for genes whose change of expression during the cell cycle is likely, lamin C, beta-actin, alpha- and beta-tubulin, c-myc, c-fos, p53. The cDNA sequences for genes of group (a) were found to be significantly enriched in DNA of the S-phase library indicating that the cell cycle phase-specific patterns of the respective mRNA levels are conserved in the sublibraries. Sequences belonging to group (b) were also found to be enriched in DNA isolated from the sublibraries: c-fos in G1 phase, lamin C, beta-actin, tubulins, c-myc in S phase, and p53 in G1/S phase. The unexpected prevalence of c-myc and alpha-tubulin in the S-phase library is supported by Northern analysis of RNA from phase-synchronous cells. Non-phase-specific, randomly chosen sequences hybridized equally strong with DNA isolated from the different sublibraries. No significant changes of the patterns of hybridization signals were observed with DNA from different amplifications of the sublibraries when analyzed with the same DNA probe indicating that the cDNA complexities are well conserved during amplifications. Consequently, the sublibraries are useful to obtain information about the cell cycle phase-specific expression of mRNAs for other genes of interest. Since the sublibraries reflect mRNA levels of the cells growing in vivo they supply data on the physiological in vivo pattern of gene expression undisturbed by potentially unphysiological in vitro conditions.

Animals↗

Cubic phases of lipid-containing systems. The structure of phase Q223 (space group Pm3n). An X-ray scattering study.

The hexagonal (H) and the cubic (Q223) phases of the systems dodecyltrimethylammonium chloride-water and palmitoyllysophosphatidy choline-water have been studied by X-ray scattering techniques. The signs of the reflections of phase H were determined by a systematic study as a function of the water content, those of phase Q223 were assessed using a pattern recognition approach based upon the axiom that the histograms of the electron density maps of phases Q223 and H, extrapolated to the same concentration and properly normalized in scale and shape, are very similar to each other. In the case of phase Q223, all the sign combinations (the phi-sets) compatible with the observed reflections were generated, and each of the corresponding histograms was compared with the histogram of the map of phase H. One novelty of this work is the use of a highly sensitive criterion to estimate the similarity of the histograms, namely the distance in the six-dimensional space of the moments [mean value of (delta rho)n]1/n, for 3 greater than or equal to n greater than or equal to 8. In the two systems, the use of this criterion has led to the unambiguous choice of one electron density map. The maps show that the structure of phase Q223 consists of disjointed micelles (of type I), belonging to two different classes: those of one class are quasi-spherical in shape and are centered at the points a, those of the other class are disc-shaped and are centred at the points c. The results of this work rule out a structure formed by a cage-like distribution of rods enclosing a set of quasi-spherical micelles and is consistent with previous proposals. This is the second example, after that of phase Q227, of a micellar cubic phases in lipid-containing systems; all the known examples of phase Q223 are of type I, those of phase Q227 of type II.

Freeze Fracturing↗

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes.

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.

Fluorescence Polarization↗

Effect of phospholipids and a transmembrane peptide on the stability of the cubic phase of monoolein: implication for protein crystallization from a cubic phase.

The cubic phase of monoolein has successfully been used for crystallization of a number of membrane proteins. However, the mechanism of protein crystallization in the cubic phase is still unknown. It was hypothesized, that crystallization occurs at locally formed patches of bilayers. To get insight into the stability of the cubic phase, we investigated the effect of different phospholipids and a model transmembrane peptide on the lipid organization in mixed monoolein systems. Deuterium-labeled 1-oleoyl-rac-[(2)H(5)]-glycerol was used as a selective probe for (2)H NMR. The phase behavior of the phospholipids was followed by (31)P NMR. Upon incorporation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidic acid, the cubic phase of monoolein transformed into the L(alpha) or H(II) phase depending on the phase preference of the phospholipid and its concentration. The ability of phospholipids to destabilize the cubic phase was found to be dependent on the phospholipid packing properties. Electrostatic repulsion facilitated the cubic-to-L(alpha) transition. Incorporation of the transmembrane peptide KALP31 induced formation of the L(alpha) phase with tightly packed lipid molecules. In all cases when phase separation occurs, monoolein and phospholipid participate in both phases. The implications of these findings for protein crystallization are discussed.

Crystallization↗

Forced coalescence phasing: a method for ab initio determination of crystallographic phases.

A method has been developed for ab initio determination of crystallographic phases. This technique, called forced coalescence phasing (FCP), is implemented on a computer and uses an automated iterative procedure that combines real space filtering with numerically seeded Fourier transforms to solve the crystallographic phase problem. This approach is fundamentally different from that of traditional direct methods of phasing, which rely on structure invariant probabilistic phase relationships. In FCP, the process begins with an appropriate set of atoms randomly distributed throughout the unit cell. In subsequent cycles of the program, these atoms undergo continual rearrangements ultimately forming the correct molecular structure(s) consistent with the observed x-ray data. In each cycle, the molecular rearrangement is directed by an electron density (Fourier) map calculated using specially formulated numerical seed coefficients that, along with the phase angles for the map, are derived from the arrangement of atoms in the preceding cycle. The method has been tested using actual x-ray data from three organic compounds. For each data set, 100 separate phase determination trials were conducted, each trial beginning with a different set of randomly generated starting phases. Correct phase sets were successfully determined in all of the trials with most trials requiring fewer than 50 cycles of the FCP program. In addition to its effectiveness in small molecule phase determination, FCP offers unexplored potential in the application of real-space methods to ab initio phasing of proteins and other macromolecule structures.

Journal Article↗

An improved phase-extension procedure for isomorphous replacement phases.

A new phase-extension procedure has been applied to isomorphous replacement data and shown to yield improved phases and maps compared with standard solvent flattening operating on a full set of centroid phases. In this procedure, a starting subset of core phases is selected based on the sharpness of the phase-probability curves. Phase extension using solvent flattening as the density-modification procedure is then carried out, gradually adding additional phases. In tests with known protein structures, the mean phase errors for the output expanded phase sets were reduced by 3-9 degrees and the corresponding map correlation coefficients were increased by 0.05-0.18 relative to phase sets from standard solvent-flattening procedures. With SIR data, the lowest final mean phase errors were approximately 58 degrees and the corresponding map correlation coefficients were in the range 0.53-0.68.

Protein Conformation↗

Fate of phase I and phase II Coxiella burnetii in several macrophage-like tumor cell lines.

Several macrophage-like tumor cell lines of murine origin were exposed to phase I and phase II Coxiella burnetii, and the subsequent fate of the parasites was determined by electron and bright-field microscopy. Phase I C. burnetii proliferated within and established a persistent infection of P388D1, J774, and PU-5-IR cell lines but not of WEHI-3 and WEHI-274 cell lines. Phase II C. burnetii, however, entered into and persistently infected all five cell lines. The parasites proliferated within vacuoles. Macrophage cell lines persistently infected with phase I and phase II C. burnetii were maintained for over 200 and 100 days, respectively. Within P388D1 cells, the phase I C. burnetii converted, in part, to phase II; phase II organisms remained in the phase II state. The differential fate of the two rickettsial phases after exposure to the WEHI-3 and WEHI-274 cells may be attributable to surface differences such as lipopolysaccharide content.

Animals↗

Two-phase helical CT for pancreatic tumors: pancreatic versus hepatic phase enhancement of tumor, pancreas, and vascular structures.

PURPOSE: To quantitatively evaluate and validate a two-phase helical computed tomographic (CT) protocol for evaluation of pancreatic tumors. MATERIALS AND METHODS: Twenty-seven patients with pathologically proved pancreatic adenocarcinomas prospectively underwent two-phase CT examination with helical acquisition during the pancreatic phase (40-70 seconds after infusion of intravenous contrast material at 3 mL/sec) and the hepatic phase (70-100 seconds after infusion). Mean CT attenuation values of tumor, bordering pancreas, and all major peripancreatic vessels were obtained for both time intervals. RESULTS: Mean tumor-pancreas contrast was significantly greater during the pancreatic phase (67 HU +/- 19) than the hepatic phase (39 HU +/- 16) (P < .001) This was the result of both greater enhancement of normal pancreas and lower tumor enhancement during the pancreatic phase. Opacification of all vascular structures, including the portal vein, was also greater during the pancreatic phase (P < .001). CONCLUSION: Two-phase helical CT with pancreatic phase acquisition provides statistically significantly better pancreatic, arterial, and portal venous enhancement than that of hepatic phase imaging, with improved tumor-pancreas contrast.

Adenocarcinoma↗

Biomechanical properties of duodenal wall and duodenal tone during phase I and phase II of the MMC.

We used a new method, impedance planimetry, to look at variations in compliance, tone, and distension-induced peristaltic activity during phase I and phase II of the migrating myoelectric complex (MMC) in the human duodenum. A balloon was inflated stepwise with pressures up to 30 cmH2O in the duodenum, while the pressure and balloon cross-sectional area (CSA) were measured simultaneously. The biomechanical wall parameters were calculated from these measurements. Nine duodenal phase IIIs were recorded in six subjects. A balloon pressure of 20 cmH2O induced a smaller CSA in early phase I [266 (236-324) mm2] than in late phase II [385 (276-474) mm2] (P < 0.05). Balloon distensions elicited no contractions in phase I, whereas they increased contractile activity 60% (P < 0.05) proximal to the balloon and 4% distal to the balloon in late phase II. Step distensions in phase I with balloon pressures between 10 and 30 cmH2O increased the CSA from 40 (30-81) to 645 (603-704) mm2. It increased circumferential wall tension from 35 (28-63) to 429 (402-466) mm x cmH2O and the pressure elastic modulus from 9.7 (9.0-14.7) to 33.8 (27.6-33.8) cmH2O, respectively. Thus compliance differs from phase I to phase II. This is most likely caused by increased smooth muscle tone during phase I. Duodenal wall stiffness increases with the balloon pressure applied.

Adult↗

Photic phase response curve in Octodon degus: assessment as a function of activity phase preference.

Light exposure during the early and late subjective night generally phase delays and advances circadian rhythms, respectively. However, this generality was recently questioned in a photic entrainment study in Octodon degus. Because degus can invert their activity phase preference from diurnal to nocturnal as a function of activity level, assessment of phase preference is critical for computations of phase reference [circadian time (CT) 0] toward the development of a photic phase response curve. After determining activity phase preference in a 24-h light-dark cycle (LD 12:12), degus were released in constant darkness. In this study, diurnal (n = 5) and nocturnal (n = 7) degus were randomly subjected to 1-h light pulses (30-35 lx) at many circadian phases (CT 1-6: n = 7; CT 7-12: n = 8; CT 13-18: n = 8; and CT 19-24: n = 7). The circadian phase of body temperature (Tb) onset was defined as CT 12 in nocturnal animals. In diurnal animals, CT 0 was determined as Tb onset + 1 h. Light phase delayed and advanced circadian rhythms when delivered during the early (CT 13-16) and late (CT 20-23) subjective night, respectively. No significant phase shifts were observed during the middle of the subjective day (CT 3-10). Thus, regardless of activity phase preference, photic entrainment of the circadian pacemaker in Octodon degus is similar to most other diurnal and nocturnal species, suggesting that entrainment mechanisms do not determine overt diurnal and nocturnal behavior.

Activity Cycles↗

Comparison of temperature-phased and two-phase anaerobic co-digestion of primary sludge and municipal solid waste.

Characterization of the similarities and differences between two-phase and temperature-phased systems treating primary wastewater sludge (PS) and the organic fraction of municipal solid waste (OFMSW) as substrate was performed by comparing the rates of key steps, including hydrolysis and methanogenesis. Aceticlastic methanogenic rates were determined using batch respirometric tests with inocula from operating two-phase and temperature-phased systems. The initial methane production rates ranged from 0.32 to 0.93 mL methane/g volatile solids (VS)h for all systems, with the greatest rates observed from the first stage of the temperature-phased system. Hydrolysis rates were determined from particulate chemical oxygen demand destruction. The first stage of the temperature-phased system had greater specific hydrolysis rates than the first stage of the two-phase system at each operating condition. The temperature-phased system outperformed the two-phase system in terms of methane production and VS destruction when treating a mixed OFMSW-PS stream at OFMSW-to-PS ratios of 0:100, 20:80, and 40:60. When the feed ratios were 60:40 and 80:20 OFMSW-PS, there was no significant difference in the performance of the two systems. The overall methane yield and VS destruction of the temperature-phased system ranged from 0.299 to 0.418 L/g VS fed and 47.5 to 71.6%, respectively. The overall methane yield and VS destruction of the two-phase system ranged from 0.281 to 0.332 L/g VS fed and 39.6 to 69.3%, respectively.

Bacteria, Anaerobic↗

Phase-inversion sonography during the liver-specific late phase of contrast enhancement: improved detection of liver metastases.

OBJECTIVE: The purpose of our study was to assess whether phase-inversion sonography during the late, liver-specific phase of contrast enhancement using Levovist improves the detection of hepatic metastases relative to unenhanced conventional B-mode sonography. SUBJECTS AND METHODS: Sixty-two patients were studied with unenhanced B-mode sonography and phase-inversion sonography 2.5 min after the injection of Levovist. All patients underwent one reference examination (CT, MR imaging, or intraoperative sonography). The conspicuity, number, size, and distribution of metastases before and after contrast administration as judged by a sonographer (who was unaware of other imaging findings) were compared with each other and with reference imaging. RESULTS: The conspicuity of metastases was improved by contrast-enhanced phase inversion in 94% of patients. Thirty-nine patients showed metastases on reference imaging; 36 of these were positive on baseline sonography and 38 on phase-inversion sonography. Phase-inversion sonography showed more reference imaging-confirmed metastases than baseline sonography in 28 patients (45%). The average number of confirmed metastases per patient was 3.06 for baseline sonography and 5.42 for contrast-enhanced phase-inversion sonography (p < 0.01). The average sensitivity for detecting individual metastases improved from 63% to 91%. Metastases of less than 1 cm were shown in 14 patients on baseline sonography, in 24 patients on phase-inversion sonography, and in 26 on reference imaging. Both sonographic techniques showed false-positive lesions in six patients. CONCLUSION: Contrast-enhanced phase-inversion sonography in the liver-specific phase of contrast enhancement using Levovist provides a marked improvement in the detection of hepatic metastases relative to unenhanced conventional sonography, without loss of specificity. Phase-inversion sonography was particularly advantageous in detecting small metastases and may be a competitive alternative to CT and MR imaging.

Adult↗

Coordinated regulation of M phase exit and S phase entry by the Cdc2 activity level in the early embryonic cell cycle.

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.

Animals↗

Pediatric phase I drug tolerance: a review and comparison of recent adult and pediatric phase I trials.

PURPOSE: We evaluated the ratio of pediatric to adult maximum tolerated doses (MTDs) from 70 Phase I studies conducted between 1975 and 1995. The aim of this study was to determine whether previously observed differences in drug tolerance between adult and pediatric Phase I patients have persisted over the 20-year period of this analysis. PATIENTS AND METHODS: Phase I trials of pediatric and adult patients with solid tumors as the predominant diagnosis and sharing similar dosing regimens were evaluated. For consistent comparison between Phase I studies, the MTD was defined as the drug dose one level below that yielding dose-limiting toxicity in >30% of patients. The ratio of pediatric to adult MTDs was calculated and plotted chronologically by year of pediatric study closure. Statistical evaluation of MTD ratios included regression and correlation analysis. The extent of therapy before Phase I study entry was also examined. RESULTS: Ninety-three Phase I studies were reviewed. Twenty-one drugs (70 studies) met our criteria for paired review of MTDs and analysis of the variation of ratio with time. The pediatric to adult MTD ratios ranged from 0.4 to 2.8, with a median of 1.2. Regression analysis of the ratio of MTD versus date of pediatric study closure supports a linear relationship of decreasing ratio with time (p<0.01). Analysis of the regression line predicts MTD ratios of 2.02 and 0.76 for 1974 and 1995, respectively. Of patients included in this analysis, 37.1% and 68.6% of adult and pediatric patients, respectively, were considered to have been heavily pretreated before study entry. A significant (p<0.001) downward trend with time was observed in the proportion of adult patients entering Phase I studies who had received both radiation and chemotherapy. CONCLUSIONS: The results of this review continue to show an equal or greater drug tolerance in the pediatric population when compared with adult patients for most drugs studied during Phase I trials. However, there appears to be significant trend of decreasing differences in drug tolerance between pediatric and adult Phase I patients with time, as defined by the descent of the MTD ratio toward values <1.0. Mechanisms to explain greater drug tolerance in children and the observation of decreasing maximum tolerated dose ratios with time are discussed. Limited data suggest that changes in degree of therapy before Phase I study entry may be influencing the MTD ratio.

Adult↗

Multistep phase difference phase contrast imaging.

A new technique for multistep phase-contrast image processing is presented. The N-step method consists of simply forming the linear average of the N-1 adjacent phase-difference signals. It has similar noise reduction properties as other multistep techniques, but the simplicity of the noise variance of the N-step technique allows intuitive insight into phase-difference phase-contrast processing and noise reduction, which can aid in the design of efficient and improved phase-contrast imaging sequences. As well, the computational simplicity of the N-step phase-difference technique compared with any other known multistep technique is advantageous. Like other multistep techniques, it has far more efficient noise reduction properties than simple two-step, multiple average phase-contrast imaging, even when normalized for total scan time. A three-step phase-difference velocity image has 50% less variance than an image acquired with two steps and two scans averaged but is obtained in 25% less scan time. Given its advantages, it should now be the chosen technique for increasing velocity-to-noise and contrast-to-noise ratios in all phase-difference phase-contrast clinical applications.

Blood Flow Velocity↗

Signal-to-noise in phase angle reconstruction: dynamic range extension using phase reference offsets.

The dynamic range of phase-reconstructed magnetic resonance images is compared to that of magnitude-reconstructed images. From analysis of propagation of errors, the phase angle noise is phase-independent and given in radians by sigma ([I])/[I], the noise-to-signal ratio of the corresponding magnitude-reconstructed image. As the phase can range from minus pi to pi, the phase angle dynamic range is 2 pi times that of the signal magnitude. These results agree with experiment, verifying that the noise in the two receiver channels is uncorrelated. An artifact-free technique is presented for correcting phase spillover, which further extends the phase angle dynamic range. The reconstruction-based reference phase is adjusted on a local basis so that the boundary of phase wraparound is reconstructed near the center of the [- pi, pi] interval. For a particular flow study, the phase signal-to-noise was extended over twofold by spillover correction, to a value 15 times that of the magnitude signal-to-noise.

Algorithms↗

Artifacts introduced by zero order phase correction in proton NMR spectroscopy and a method of elimination by phase filtering.

In in-vivo applications of proton NMR spectroscopic imaging, an oscillatory "ringing" artifact has been observed in some of the spectra. The source of this artifact was found to be the presence of a harmonic "beating" effect in the amplitude of the water reference free induction decay (FID) which was used for zero order phase correction for B0 inhomogeneity and eddy current compensation. The source of the beats was found to be the presence of distinct populations of spins resonating at slightly different frequencies. When the common method of zero order phase correction was implemented using such an FID, the resulting phase-corrected, water-suppressed spectra displayed ringing. Examination of the unwrapped phase correction angle revealed unexpected jumps in phase at points in time corresponding to nodes in the amplitude of the FID. Low-pass filtering of the phase correction angle of the reference FID was found to smooth out these unanticipated phase jumps. When used as a reference for phase correction, the filtered phase information gave a phase-corrected, water-suppressed spectrum free from ringing.

Artifacts↗

Late-phase enhancement of the upstream portion of pancreatic adenocarcinoma on dual-phase helical CT.

BACKGROUND: Late-phase enhancement of pancreatic parenchyma upstream (tail side) of pancreatic adenocarcinoma is found frequently on dual-phase helical computed tomography (CT). We measured the frequency of late-phase enhancement of the upstream portion of pancreatic adenocarcinoma and normal pancreatic parenchyma using dual-phase helical CT. METHODS: Twenty-one patients with pancreatic adenocarcinoma and nontumorous pancreas upstream of tumors were compared with 100 control patients without pancreatic disease. Early and late scans started at 25 and 75 s, respectively, after intravenous injection of contrast material. The attenuation values of normal and nontumorous pancreas upstream of tumors were assessed in three phases: precontrast, early, and late enhanced. Enhancement ratio (ER) was calculated as ER = (late phase - precontrast)/(early phase - precontrast). RESULTS: Late-phase enhancements (ER > 1.0) were seen in 86% of upstream pancreas and 10% of normal pancreas. The mean ER of upstream pancreas was significantly higher than that of normal pancreas (p < 0.01). CONCLUSION: Late-phase enhancement of the pancreas upstream of the tumor is frequently observed in patients with pancreatic adenocarcinoma. Late-phase enhancement and histology showed a correlation for chronic obstructing pancreatitis in five patients.

Adenocarcinoma↗