Robotic Mammosphere Assay for High-Throughput Screening in Triple-Negative Breast Cancer.

In order to identify novel treatment principles specifically affecting cancer stem cells in triple-negative breast cancer, we have developed a high-throughput screening method based on the mammosphere and anoikis resistance assays allowing us to screen compounds using a functional readout. The assay was validated against manual protocols and through the use of positive controls, such as the response to hypoxia and treatment with the known cancer stem cell-targeting compound salinomycin. Manual and robotic procedures were compared and produced similar results in cell handling, cell cultures, and counting techniques, with no statistically significant difference produced from either method. The variance between samples processed manually versus robotically was no greater than 0.012, while Levene's test of significance was 0.2, indicating no significant difference between mammosphere data produced manually or robotically. Through the screening of 989 FDA-approved drugs and a follow-up screen assessing the antineoplastic subgroup, we have identified three therapeutic compounds with the ability to modulate the breast cancer stem cell fraction in the triple-negative breast cancer cell line MDA-MB-231, highlighting their potential usage as stem cell-specific adjuvant treatments.

A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.

Retroaortic left renal vein and its implications in abdominal aortic surgery.

The embryological development of the retroperitoneal venous system is a complex process. As a result, the anatomy of the inferior vena cava (IVC) and renal veins shows extensive variability. Improper completion of this process may lead to six anatomical variants: retroaortic left renal vein (LRV) types I and II, circumaortic venous collar, duplication of the IVC, transposition or left-sided IVC, and preaortic iliac confluence. All six are infrequent, but may be encountered during abdominal aortic reconstruction and pose challenging problems to the operating surgeon. Failure to appreciate these anomalies can lead to inadvertent injury and major venous bleeding. Preoperative diagnosis can be made on a CT scan, but this is not always performed prior to aortic surgery. In this report, we analyze two cases of retroaortic LRV complicating abdominal aortic aneurysmectomy, describe the most common infrarenal venous anomalies encountered during aortic surgery, and briefly review the literature.

Rupture of the abdominal aorta in patients with Ehlers-Danlos syndrome.

Ehlers-Danlos syndrome (EDS) is a heterogeneous inherited disorder of collagen synthesis. Type IV is frequently associated with major vascular catastrophes and challenges the vascular surgeon with its varied clinical presentation and the difficulty of vascular repair. Rupture of the abdominal aorta is one of the most serious complications and is associated with nearly 100% mortality rate. We describe here three patients with type IV EDS.

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

False aneurysm of the popliteal artery: a rare complication of total knee replacement.

We describe a 72-year-old woman with a false aneurysm of the mid-popliteal artery after a total knee replacement, presenting with a pulsatile swelling and an audible bruit in the popliteal fossa. The diagnosis was confirmed by duplex ultrasound. Surgical repair was undertaken using a saphenous vein patch to reconstruct the arterial defect. A false aneurysm of the popliteal artery following total knee replacement is an extremely rare occurrence. To our knowledge, only four other cases have been reported in the English-language literature.

Abdominal aortic aneurysm: the role of clinical examination and opportunistic detection.

OBJECTIVES: to investigate the method of discovery of abdominal aortic aneurysms (AAA) in a district general hospital setting. DESIGN: retrospective study. MATERIALS AND METHODS: we analysed 198 patients with an AAA who presented to our unit over a 3-year period. The method of initial diagnosis, size of the AAA and whether this was palpable, irrespective of the method of detection, were recorded. RESULTS: ninety-five (48%) were discovered clinically, 74 (37.4%) during a radiological investigation, and 29 (14.6%) at laparotomy. Of the 74 AAAs first detected radiologically, subsequent physical examination showed that 28 (37.8%) were in fact palpable and missed at presentation. The average size of those discovered clinically (6. 48+/-1.32 cm) was larger than those found radiologically (5.37+/-1. 44 cm, p<0.001) or at operation (5.43+/-1.48 cm, p=0.039). The average diameter of the palpable AAAs was also greater than that of the non-palpable AAAs (6.42+/-1.24 cm vs. 4.86+/-1.38 cm, p<0.001). CONCLUSIONS: opportunistic detection of a clinically unsuspected aneurysm during clinical examination or investigation for another reason is the most common way the diagnosis of an AAA is made. Almost half of the aneurysms were diagnosed clinically, but physical examination also missed more than a third of those detected radiologically. Despite technological advancement, clinical examination still plays a paramount role in the detection of AAAs. Larger AAAs are usually palpable and more likely to be detected on clinical examination.

Conserved residues in the mechanism of the E. coli Class II FBP-aldolase.

The two classes of fructose-1,6-bisphosphate aldolase both catalyse the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The Class I aldolases use Schiff base formation as part of their catalytic mechanism, whereas the Class II enzymes are zinc-containing metalloproteins. The mechanism of the Class II enzymes is less well understood than their Class I counterparts. We have combined sequence alignments of the Class II family of enzymes with examination of the crystal structure of the enzyme to highlight potentially important aspartate and asparagine residues in the enzyme mechanism. Asp109, Asp144, Asp288, Asp290, Asp329 and Asn286 were targeted for site-directed mutagenesis and the resulting proteins purified and characterised by steady-state kinetics using either a coupled assay system to study the overall cleavage reaction or using the hexacyanoferrate (III) oxidation of the enzyme bound intermediate carbanion to investigate partial reactions. The results showed only minor changes in the kinetic parameters for the Asp144, Asp288, Asp290 and Asp329 mutants, suggesting that these residues play only minor or indirect roles in catalysis. By contrast, mutation of Asp109 or Asn286 caused 3000-fold and 8000-fold decreases in the kcat of the reaction, respectively. Coupled with the kinetics measured for the partial reactions the results clearly demonstrate a role for Asn286 in catalysis and in binding the ketonic end of the substrate. Fourier transform infra-red spectroscopy of the wild-type and mutant enzymes has further delineated the role of Asp109 as being critically involved in the polarisation of the carbonyl group of glyceraldehyde 3-phosphate.

The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase.

The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.

Buccal glands of adults of the lamprey Mordacia mordax, including comparisons with other species.

Mordacia mordax is one of the two anadromous parasitic lamprey species of the southern hemisphere family Mordaciidae. Its adults possess two lateral buccal glands and one central buccal gland. When the tongue-like piston is retracted, the buccal glands occupy much of the opening of the oral cavity at the rear of the buccal cavity. The glands contain numerous tube-like, ductless secretory units, which discharge directly into the buccal cavity. Their secretory epithelial cells contain numerous granules, some of which are zymogen-like, while others have a beaded, spiralled appearance. The similarity of the latter to mast cell granules suggests that they may likewise produce an anticoagulant, which would be valuable to a presumed blood feeder such as M. mordax. The mucus produced by these cells could act as a carrier for the secretions and as an adhesive for promoting retention of t he secretions on the host's surface. When the young adults is transferred to salt water, the buccal glands increase their production and discharge of secretions. Since the glands are not enclosed in musculature, their secretions are probably discharged by mechanical pressure applied by the forward movement of the head of the tooth-bearing piston into the buccal cavity. An account is given of the way in which the location, number, glandular organization, secretory granules, and type of secretion of the buccal glands of M. mordax, and thus presumably also their mode of function, differ markedly from those of members of the other lamprey family found in the southern hemisphere, and of all holarrctic lampreys. © 1995 Wiley-Liss, Inc.

The reaction of GroEL (cpn 60) with the ATP analogue 2',3' dialdehyde ATP.

The reaction of the E. coli chaperonin GroEL (cpn 60) with the ATP analogue 2',3' oxidised ATP (oATP) has been studied. Treatment with the reagent leads to loss of the ATPase activity of GroEL in a pseudo-first-order fashion; this can be prevented by inclusion of ATP in the reaction mixture. Measurements of the stoichiometry of the reaction indicate that the loss of activity corresponds to the incorporation of about one oATP per subunit of GroEL. From analysis of the sequences of modified peptides it is proposed that the reaction probably occurs with one or both of the two cysteines Cys-457 and Cys-518, although the instability of the adduct(s) makes a definite identification of the site(s) of reaction difficult. The involvement of Cys side chains in the reaction with oATP was confirmed by using Nbs2 (5,5'-dithiobis(2-nitrobenzoate)) to estimate thiol groups in both modified and unmodified GroEL.

The unfolding and attempted refolding of the bacterial chaperone protein groEL (cpn60).

The unfolding of the bacterial chaperone protein groEL (cpn60) in solutions of guanidinium chloride (GdnHCl) has been studied. From the results of CD, fluorescence and light scattering, it is clear that major structural transitions in the protein occur over the range 1.0-1.5 M GdnHCl. The ATPase activity of the protein is lost at lower concentrations (0.75 M). After denaturation in concentrations of GdnHCl above 1.5 M, removal of the denaturing agent by dialysis results in very nearly complete regain of secondary structure (as judged by CD), but not the regain of correct tertiary or quaternary structure, or ATPase activity. The product was shown to be very sensitive to proteolysis by thermolysin, unlike the native protein, and not to show enhanced binding of ANS, a characteristic property of the 'molten globule' state of proteins. The results are discussed in relation to current information concerning the assembly of the groEL protein.

The response of rapidly formed adult human endothelial-cell monolayers to shear stress of flow: a comparison of fibronectin-coated Teflon and gelatin-impregnated Dacron grafts.

Endothelial cells labeled with indium 111-oxine were seeded in supraconfluent densities onto fibronectin-coated expanded polytetrafluoroethylene (ePTFE) and gelatin-impregnated Dacron graft segments. These grafts with rapidly formed endothelial-cell monolayers were then exposed to varying shear stresses at flow rates of 200 and 300 ml/min, using tissue culture medium in an artificial flow circuit. As the loss of radioactivity represented endothelial-cell loss, cell retention was calculated by the ratio of counts recorded at different time points during 2 hours of flow to initial counts. Although initial cell adherence to gelatin-impregnated Dacron graft segments was poor compared to ePTFE, once cells were attached they resisted shear stress of flow better at 200 ml/min and equally well at 300 ml/min. The cell retention on fibronectin-coated ePTFE was 55.4 +/- 12.9% at 200 ml/min and 56.5 +/- 15.2% at 300 ml/min; cell retention for gelatin-impregnated Dacron graft segments was 69.0 +/- 6.0% and 66.5 +/- 5.5%, respectively. Qualitatively scanning electron microscopy of both ePTFE and gelatin-impregnated Dacron graft segments showed patchy coverage of grafts with cells. There was preferential attachment of endothelial cells to the nodes on ePTFE, although on gelatin-impregnated Dacron graft segments, cells conformed to the Dacron fibers at different levels and directions with evidence of bridging in the gaps between individual fibers. This study shows conclusively that rapidly formed endothelial-cell monolayers on ePTFE and gelatin-impregnated Dacron graft segments resisted a shear stress of flow equal to that seen in a femoropopliteal vein graft with significant cell retention at 2 hours.

Spinal cord monitoring in patients with nonidiopathic spinal deformities using somatosensory evoked potentials.

Seventy-nine somatosensory evoked potentials were intraoperatively recorded in 52 patients undergoing spinal surgery for nonidiopathic spinal deformities. There were 37 true-negative, 28 true-positive (a significant change in the somatosensory evoked potential related to the surgical process), and 14 false-positive (a significant change in the somatosensory evoked potential not related to a surgical event) readings. There were, however, no postoperative neurologic deficits with any of the true-positive readings and no false negatives. Spinal and subcortical somatosensory evoked potentials gave few false-positive readings. True-positive somatosensory evoked potentials occurred in 44% of the patients with neuromuscular deformities, 17% with congenital deformities, 45% with Luque instrumentation, 22% with Harrington instrumentation, and none with fusion in situ. Fifty percent of the true positives occurred while the sublaminar wires were tightened. The predictive accuracy of intraoperative spinal cord monitoring in this patient population is not high, but the sensitivity to potentially harmful surgical events is high.

Comparison of different vascular prostheses and matrices in relation to endothelial seeding.

Thin-walled expanded polytetrafluoroethylene (ePTFE), woven Dacron and gelatin-impregnated Dacron (Gelseal) vascular grafts were compared, the grafts being coated with three different matrices: collagen IV, fibronectin and preclot matrix. In addition, untreated ePTFE and Gelseal were examined. The graft segments, coated with these matrices, were incubated with radiolabelled adult human endothelial cells for 30, 60 and 90 min. Endothelial cell adherence was calculated from the ratio of radioactive counts in the grafts to counts in grafts plus supernatants. Endothelial cell attachment to untreated grafts was poor, but a suitable matrix significantly improved adherence. All three matrices tested gave good results, although preclot was best; 30-60 min incubation was sufficient for optimum cell attachment. Cell adherence to both Dacron and ePTFE was significantly better than to Gelseal. The type of prosthetic polymer and the substrate protein coating used to promote endothelial cell adherence are two important factors which may determine the ultimate success of endothelial seeding in the operating room.