Bacteriorhodopsin crystal growth in reduced gravity--results under the conditions, given in CPCF on board of a Space Shuttle, versus the conditions, given in DCAM on board of the Space Station Mir.

For the purpose of bio-electronics, bacteriorhodopsin was crystallized into two habits through liquid-liquid-diffusion, namely individual needles of up to 1.9 mm in length and needle bunch-like clusters of up 4.9 mm in total length. In both the reduced gravity experiments performed, the morphology of the individual needles (crystal form A) had improved in terms of sharp needle edges and compact needle packing, compared to the parallel ground controls. For the long duration wide range low gravity condition in the "Diffusion-controlled Crystallization Apparatus for Microgravity (DCAM)" on Mir (STS-89 up), needle bunches on average were longer there than on the ground, while the compactness of the clusters, i.e. the average ratio of clustered length to clustered width was the reverse. Some exceptionally large individuals needles were grown in DCAM. For the "Commercial Protein Crystallization Facility (CPCF)" in short duration high definition microgravity conditions during a science mission of the Space Shuttle Discovery (STS-95), size and shape of the individual needles were homogeneous and superior to those of both the parallel ground controls and the results in DCAM. In CPCF, the average volume of the individual needles in suspension was increased by 50% in microgravity compared to those in the ground control.

Five recombinant fragments of human serum albumin-tools for the characterization of the warfarin binding site.

Human serum albumin (HSA) interacts with a vast array of chemically diverse ligands at specific binding sites. To pinpoint the essential structural elements for the formation of the warfarin binding site on human serum albumin, a defined set of five recombinant proteins comprising combinations of domains and/or subdomains of the N-terminal part were prepared and characterized by biochemical standard procedures, tryptophanyl fluorescence, and circular dichroic measurements, indicating well-preserved secondary and tertiary structures. Affinity constants for binding to warfarin were estimated by fluorescence titration experiments and found to be highest for HSA-DOM I-II and HSA, followed by HSA-DOM IB-II, HSA-DOM II, and HSA-DOM I-IIA. In addition, ultraviolet difference spectroscopy and induced circular dichroism experiments were carried out to get an in depth understanding of the binding mechanism of warfarin to the fragments as stand-alone proteins. This systematic study indicates that the primary warfarin binding site is centered in subdomain IIA with indispensable structural contributions of subdomain IIB and domain I, while domain III is not involved in this binding site, underlining the great potential that lies in the use of combinations of recombinant fragments for the study and accurate localization of ligand binding sites on HSA.

Conformational transitions of the three recombinant domains of human serum albumin depending on pH.

Human serum albumin (HSA) is a protein of 66.5 kDa that is composed of three homologous domains, each of which displays specific structural and functional characteristics. HSA is known to undergo different pH-dependent structural transitions, the N-F and F-E transitions in the acid pH region and the N-B transition at slightly alkaline pH. In order to elucidate the structural behavior of the recombinant HSA domains as stand-alone proteins and to investigate the molecular and structural origins of the pH-induced conformational changes of the intact molecule, we have employed fluorescence and circular dichroic methods. Here we provide evidence that the loosening of the HSA structure in the N-F transition takes place primarily in HSA-DOM III and that HSA-DOM I undergoes a structural rearrangement with only minor changes in secondary structure, whereas HSA-DOM II transforms to a molten globule-like state as the pH is reduced. In the pH region of the N-B transition of HSA, HSA-DOM I and HSA-DOM II experience a tertiary structural isomerization, whereas with HSA-DOM III no alterations in tertiary structure are observed, as judged from near-UV CD and fluorescence measurements.

The three recombinant domains of human serum albumin. Structural characterization and ligand binding properties.

In an attempt to systematically dissect the ligand binding properties of human serum albumin (HSA), the gene segments encoding each of its three domains were defined based on their conserved homologous structural motifs and separately cloned into a secretion vector for Pichia pastoris. We were able to establish a generally applicable purification protocol based on Cibacron Blue affinity chromatography, suggesting that each of the three domains carries a binding site specific for this ligand. Proteins were characterized by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, gel filtration, N-terminal sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as well as near- and far-UV CD. In addition to the affinity chromatography ligand Cibacron Blue, binding properties toward hemin, warfarin, and diazepam, each of which represents a standard ligand for HSA, respectively, were investigated by the measurement of induced circular dichroism. Clear experimental evidence is provided here for the location of the primary hemin binding site to be on domain I of HSA, and for the primary diazepam binding site to be on domain III. Further, secondary binding sites were found for hemin to be located on domains II and III, and for diazepam on domain I. The warfarin binding site was located primarily on domain II, while on domain I, a secondary binding site and/or parts of the primary binding site were found.

Laparoscopy with laparoscopic ultrasonography in the TNM staging of pancreatic carcinoma.

A prospective study was performed comparing laparoscopy with laparoscopic ultrasonography (LapUS), transabdominal ultrasonography (USS), computed tomography (CT), and selective visceral angiography with portal phase venography (SVA) for the assessment of resectability in 50 patients with pancreatic or periampullary cancer. The results were stratified by TNM stages. Tumor unresectability was demonstrated in 36 patients (72%). The sensitivity of LapUS for demonstrating the index lesion was 96%. Laparoscopic ultrasonography failed to predict factors precluding resection by T stage in six patients, and there were no significant differences in the ability of any modality to predict local resectability (predictive value 58-73%). Laparoscopic ultrasonography did not overestimate T stage and was significantly more specific for assessing unresectability compared with USS (100% vs. 64%, p<0.05) and CT (100% vs. 47%, p<0.005). No imaging investigation was able to assess the N stage accurately. Metastases were confirmed in 16 patients (32%), with LapUS proving significantly more sensitive than USS (94% vs. 29%, p<0.001) and CT (94% vs. 33%, p<0.005). The addition of LapUS to the laparoscopic examination did not change the M stage in any patient, as all metastases were superficially located. Laparoscopy with LapUS was the most reliable method for assessing overall tumour resectability and was significantly more predictive than CT (97% vs. 79%, p<0.005). These results confirm that laparoscopy is indispensable for detecting occult intraabdominal metastases. LapUS reliably predicts tumor unresectability, offsetting the tendency of USS and CT to overestimate T stage. Methods of accurate N staging remain elusive, and the use of routine SVA is not justified.

Supervised surgical trainees can perform pancreatic resections safely.

With the recent changes in surgical training and sub-specialisation, the role of surgical trainees in more advanced surgical procedures has come into question. In order to examine this further, we analysed the early outcome of patients in a single surgical unit undergoing pancreatic resections, with regard to the grade of the surgeon performing the operation. Between January 1994 and May 1996, data were collected prospectively on all the patients undergoing pancreatic resections with regards to the grade of the surgeon performing the procedure and the early outcome following the operation. Sixty-two patients underwent pancreatic resections for both benign and malignant diseases. Overall, 19 operations (31%) were performed by trainees under supervision, 14 of the 40 pancreatico-duodenectomies (35%) and 5 of the 19 left partial pancreatectomies (26%). All 3 total pancreatectomies were carried out by consultants. In the 43 patients operated upon by the consultants, there were 8 anastomotic leaks (19%) and 1 death. In the 19 patients operated upon by the supervised trainees, there were only 2 anastomotic leaks (11%) and no deaths. This series has demonstrated that in a unit with a major interest and large workload in pancreatic surgery, there appears to be no difference between a consultant and a supervised trainee in the early outcome following pancreatic resections.

Cystic tumours of the pancreas.

BACKGROUND: Cystic pancreatic tumours may be misdiagnosed as pseudocysts. METHODS: From August 1990 to January 1998, 21 patients (16 women) with a median age of 60 years underwent operation for a cystic mass in the pancreas with histological confirmation of neoplasia (six serous cystadenoma (SCA), three mucinous cystic adenoma (MCA), ten mucinous cystadenocarcinoma (MCAC), one ductal adenocarcinoma with cystic degeneration, one cystic islet cell tumour). RESULTS: While the lesion had been labelled as a pseudocyst in eight patients, only one patient (MCA in the pancreatic head) had had acute pancreatitis previously. In seven patients the computed tomogram (CT) lacked suspicious neoplastic features, while endoscopic retrograde cholangiopancreatography, angiography and percutaneous cyst fluid analysis were unhelpful or misleading in 16 of 18 investigations with respect to differentiating tumour from pseudocyst. Attempted operation for cure was performed in 18 patients despite diagnostic delays of up to 6 years and initial treatment with cystenterostomy in two cases. CONCLUSION: Retrospective review revealed that all 21 cystic neoplasms could be diagnosed before operation by a history excluding previous pancreatitis (20 of 21 patients) or a CT suspicious for neoplasia (14 of 21). The diagnosis relies more on absence of previous pancreatitis and a suspicious clinician who errs on the side of resecting a pseudocyst rather than watching or draining a cystic neoplasm.

Stationary crystal diffraction with a monochromatic convergent X-ray source and application for macromolecular crystal data collection.

A diffraction geometry utilizing convergent X-rays from a polycapillary optic incident on a stationary crystal is described. A mathematical simulation of the resulting diffraction pattern (in terms of spot shape, position and intensity) is presented along with preliminary experimental results recorded from a lysozyme crystal. The effective source coverage factor is introduced to bring the reflection intensities onto the same scale. The feasibility of its application to macromolecular crystal data collection is discussed.

Glutamine-supplemented total parenteral nutrition reduces blood mononuclear cell interleukin-8 release in severe acute pancreatitis.

Glutamine, a conditionally essential amino acid, is important for immune function. It is now being formulated for incorporation into total parenteral nutrition (TPN). The aims of this study were to examine the effect of glutamine administration on lymphocyte proliferation and proinflammatory cytokine release in patients with severe acute pancreatitis. Fourteen patients were randomized (in a double-blind fashion) to receive either conventional or isocaloric, isonitrogenous glutamine-supplemented (0.22 g glutamine x kg(-1) x d(-1) as glycyl-glutamine) TPN for 7 d. DNA synthesis (index of lymphocyte proliferation) and the 24-h release of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 from peripheral blood mononuclear cells were measured in vitro on days 0, 4, and 7. Thirteen patients completed the study protocol (6 glutamine TPN, 7 conventional TPN). Glutamine supplementation increased median DNA synthesis by 3099 cpm over the study period against 219 cpm in the conventional group (increase not significantly different between the two groups) . Glutamine supplementation did not significantly influence TNF or IL-6 release, but, in contrast, median IL-8 release was reduced by day 7 in the glutamine group while it was increased in the conventional group (-17.7 ng/mL (median change over study period) versus +43.3 ng/mL, respectively; P=0.045). Small patient numbers and substantial interindividual variation limit the conclusions, but there is a trend for the glutamine group to have improved lymphocyte proliferation, and in the case of IL-8, reduced proinflammatory cytokine release.

Improved diffraction of antithrombin crystals grown in microgravity.

Crystals of antithrombin were grown both on earth and in microgravity aboard US Space Shuttle Flight STS-67. The quality of crystals grown in both environments was highly variable and many could not be indexed. The microgravity crystals, however, generally diffracted better, as demonstrated by a novel procedure that estimates the resolution of the Bragg scatter from single diffraction images, without requiring knowledge of the cell dimensions of the crystal. Whereas the best earth-grown crystals never diffracted beyond 3 angstroms resolution, the best microgravity crystal diffracted to 2.6 angstroms. The improvement, demonstrated here by a comparison of 23 microgravity and 12 earth-grown crystals, is attributed to better ordered crystal growth in microgravity, although other factors may have contributed also.

Extracolonic features of familial adenomatous polyposis in patients with sporadic colorectal cancer.

We have investigated the occurrence of attenuated extracolonic manifestations (AEMs) of familial adenomatous polyposis (FAP) in patients with non-polyposis colorectal cancer. In a prospective case-control study, we observed that significantly more colorectal cancer patients exhibited AEM than did age and sex-matched controls (19.5% vs 7.5%, P < 0.004). However patients with AEMs do not have occult FAP, as we found no heterozygous adenomatous polyposis coli (APC) gene mutations despite extensive analysis of constitutional DNA. Genome-wide DNA replication errors (RERs) occur in a proportion of colorectal cancers, particularly right-sided lesions and in almost all tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients. As AEMs have been reported in familial colon cancer cases, we investigated the relationship of AEMs to tumour RER phenotype. There was indeed an excess of AEMs in patients with right-sided tumours (30.2% of 53 patients vs 14.7% of 116 patients, P < 0.03) and in those with RER tumours (3 out of 12 patients with RER tumours vs none out of 21 patients with non-RER tumours, P < 0.05). Two patients with AEM were from HNPCC families compared with none of those without AEM (P < 0.05). The association of AEMs with colorectal cancer is intriguing, and we speculate that it may be a manifestation of mutational mosaicism of the APC gene, perhaps associated with a constitutional defect in DNA mismatch pair.

Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed to eicosapentaenoic acid in vitro.

Eicosapentaenoic acid (EPA) has been shown to have an inhibitory effect on the growth of several pancreatic cancer cell lines in vitro. This study investigates the mechanism of growth inhibition and cytotoxicity of EPA on the pancreatic cancer cell line MIA PaCa-2. Cells were analysed for cell count, viability, cell cycle distribution and ultrastructural changes. There was a time- and dose-dependent decrease in cell count and viability in cultures of pancreatic cancer cells supplemented with EPA. Flow cytometric DNA analysis of MIA PaCa-2 cells incubated with EPA demonstrated the presence of sub G1 populations corresponding to the presence of apoptotic cells and the blockade of cell cycle progression in S-phase and G2/M-phase. The presence of apoptosis in EPA-supplemented cultures was further confirmed by DNA fragmentation and ultrastructural changes associated with apoptosis. Therefore, we conclude that EPA mediates its effect on the pancreatic cancer cell line MIA PaCa-2, at least in part, via cell cycle arrest and the induction of apoptosis.

Metabolic disturbance and sensitivity to endotoxin in patients with advanced cancer: relationship to lymphocyte reactivity, tumour necrosis factor (TNF) production and survival.

Increased TNF production and impaired lymphocyte function have been individually linked with metabolic disturbance, endotoxaemia and mortality in humans. The inter-relationship between these observations was investigated in humans with cancer. In 13 patients with metastatic colorectal cancer and seven healthy volunteers, observations (n = 23) included peripheral blood mononuclear cell (PBMC) TNF production, IL-2 production and phytohaemagglutinin (PHA) response; the acute-phase protein response (APPR) (serum C-reactive protein (CRP), albumin, CRP/albumin ratio), and survival. APPR correlated with survival (CRP, r = -0.689, P = 0.006; CRP/albumin, r = -0.758, P = 0.002; albumin, r = 0.655, P = 0.011), but not with TNF production. TNF production in response to in vitro endotoxin correlated with impaired lymphocyte function in patients (r = 0.567, P = 0.043) and in the whole group (r = 0.65, P = 0.001). The ratio (basal PBMC TNF production)/(lymphocyte function) correlated with CRP (r = 0.569, P = 0.042), CRP/albumin (r = 0.617, P = 0.025), endotoxin sensitivity (r = 0.567, P = 0.043) and survival (r = -0.545, P = 0.038) in patients, and the whole group (P < 0.002). Impaired lymphocyte function may influence TNF production, endotoxin sensitivity and metabolic disturbance in humans with cancer. (r = Spearman correlation coefficient.)

Proinflammatory cytokine release by peripheral blood mononuclear cells from patients with acute pancreatitis.

Proinflammatory cytokine release was measured from peripheral blood mononuclear cells (PBMCs) isolated from six volunteers and, on admission, from 16 patients with acute pancreatitis. Tumour necrosis factor (TNF) release in patients did not differ significantly from that of volunteers, whereas both interleukin (IL) 6 and IL-8 release in patients was raised when compared with that in the volunteer group (mean(s.e.m.) IL-6 20.7(4.6) versus 9.3(1.7) ng/ml, P = 0.03; IL-8 283(40) versus 128(22) ng/ml, P = 0.04). When variation in white cell count was accounted for, IL-6 and IL-8 release but not that of TNF was significantly greater in patients with severe disease than in those with mild disease. These results point to a complex upregulation of proinflammatory cytokine release from PBMCs in patients with acute pancreatitis, components of which relate to the clinical progress of the disease.

An open-label phase I/II dose escalation study of the treatment of pancreatic cancer using lithium gammalinolenate.

There are currently no satisfactory treatments for inoperable pancreatic cancer. Median survivals for untreated patients are of the order of 100 days and, with one exception, no chemotherapy or radiotherapy regime has been found to produce a worthwhile extension of life with reasonably tolerable side effects. Gamma-linolenic acid (GLA) has been found to kill about 40 different human cancer cell lines in vitro without harming normal cells. The lithium salt of GLA (LiGLA) can be administered intravenously and a dose escalation study of a 10 day infusion followed by oral therapy in patients with inoperable pancreatic cancer was carried out in 48 patients in two centres. Peripheral venous infusion caused thrombophlebitis but this could be avoided by infusing via a central vein with appropriate heparinisation. Too rapid infusion caused haemolysis which could be avoided by slow dose escalation in the first few days and maintenance of plasma lithium below 0.8 mmol/l. Doses ranged from 7 to 77g/patient cumulatively delivered over 2-12 days. Other than the above described events there were no important side effects and patients felt well during the infusions. A Kaplan-Meier analysis showed that survival was not significantly influenced by which centre the patients were treated in, the sex of the patients or the presence or absence of histological confirmation. The presence or absence of liver metastases, the patients' Karnofsky scores and the-dose of LiGLA had significant effects on survival from treatment. A Cox proportional hazards model revealed similar results: in both centres, in both sexes, and in patients with and without liver metastases according to the model the highest doses of LiGLA were associated with longer survival times as compared with the lowest doses. LiGLA deserves investigation in a randomised prospective study.