Use and optimization of a dual-flowrate loading strategy to maximize throughput in protein-a affinity chromatography

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein-A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein-A system. A gradient-based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two-step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc-fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual-flowrate strategy was found to be superior.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

Dendritic cells from chronic lymphocytic leukemia patients are normal regardless of Ig V gene mutation status

Patients with B-type chronic lymphocytic leukemia (B-CLL) segregate into 2 subgroups based on the mutational status of the immunoglobulin (Ig) V genes and the patients in these subgroups follow very different clinical courses. To examine whether dendritic cells (DCs) generated from CLL patients can be candidates for immune therapy, we compared the phenotypic and functional capacities of DCs generated from patients of the 2 CLL subgroups (normal age-matched subjects [normal-DCs]). Our data show that immature DCs from B-CLL patients (B-CLL-DCs) have the same capacity to take up antigen as those from normal controls. Furthermore, B-CLL-DCs generated from the 2 CLL subgroups up-regulated MHC-II, CD80, CD86, CD83, CD40, and CD54 and down-regulated CD206 in response to stimulation with a cocktail of cytokines (CyC) and secreted increased levels of tumor necrosis factor alpha, interleukin (IL)-8, IL-6, IL-12 (p70), and RANTES in a manner typical of mature normal-DCs. Interestingly, CD54 was significantly more up-regulated by CyC in B-CLL-DCs compared with normal-DCs. Except for CD54, no significant differences in surface molecule expression were observed between normal-DCs and B-CLL-DCs. B-CLL-DCs from both subgroups, including 6 patients with VH1-69, that usually fare poorly, presented tetanus toxoid to autologous T cells in vitro similar to normal- DCs. Our data show that DCs generated from the B-CLL subgroup with unmutated Ig V genes are functionally normal. These results are very promising for the use of DCs from patients with poor prognosis for immunotherapy.     

QSAR study on some pyridoacridine ascididemin analogues as anti-tumor agents

Pyridoacridine ascididemin analogues have been reported as anticancer agents for their interesting antitumor activity against human cancer cells. A quantitative structure–activity relationship (QSAR) analysis of ascididemin analogues was attempted using the physicochemical parameters and the electrotopological state atom (ETSA) indices. This study indicates that the electron withdrawing substituents with higher MR (molar refractivity) value at R₁ position favor the anti-tumor activity and the presence of NHR (R is hydrogen or alkyl group) at the R₃ position has contribution to the anti-tumor activity. ETSA indices have been incorporated as independent variable in the QSAR model with physicochemical parameters. It clearly suggests the importance of atoms 2, 3, 4, 5, 6 and 7 to the anti-tumor activity.     

Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.     

Protein-tyrosine phosphatase-1B (PTP1B) mediates the anti-migratory actions of Sprouty

Mammalian Sprouty proteins have been shown to inhibit the proliferation and migration of cells in response to growth factors and serum. In this communication, using HeLa cells, we have examined the possibility that human Sprouty 2 (hSPRY2) mediates its anti-migratory actions by modulating the activity or intracellular localization of protein-tyrosine phosphatases. In HeLa cells, overexpression of hSPRY2 resulted in an increase in protein-tyrosine phosphatase (PTP1B) amount and activity in the soluble (100,000 x g) fraction of cells without an increase in total amount of cellular PTP1B. This increase in the soluble form of PTP1B was accompanied by a decrease in the amount of the enzyme in the particulate fraction. The amounts of PTP-PEST or PTP1D in the soluble fractions were not altered. Consistent with an increase in soluble PTP1B amount and activity, the tyrosine phosphorylation of cellular proteins and p130(Cas) was decreased in hSPRY2-expressing cells. In control cells, overexpression of wild-type (WT) PTP1B, but not its C215S catalytically inactive mutant mimicked the actions of hSPRY2 on tyrosine phosphorylation of cellular proteins and migration. On the other hand, in hSPRY2-expressing cells, the C215S mutant, but not WT PTP1B, increased tyrosine phosphorylation of cellular proteins and attenuated the anti-migratory actions of hSPRY2. Interestingly, neither WT nor C215S mutant forms of PTP1B modulated the anti-mitogenic actions of hSPRY2. Therefore, we conclude that an increase in soluble PTP1B activity contributes to the anti-migratory, but not anti-mitogenic, actions of hSPRY2.     

Polyphenolic Antioxidants Efficiently Protect Urease from Inactivation by Ultrasonic Cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0–6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 W/cm², 36–56°C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 W/cm², 36 or 56°C) has been characterized quantitatively, using first-order rate constants: k _in, total inactivation; k _in ^*, thermal inactivation; and k _in(us), ultrasonic inactivation. Within the range from 1 nM to 10 M, propyl gallate (PG) decreases by approximately threefold the rate of LFUS-induced inactivation of urease (56°C), whereas resorcinol poly-2-disulfide stops this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36°C) or 10 nM (56°C). At 0.2–1.0 M, human serum albumin (HSA) increases the resistance of urease treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS–HSA), formed by conjugation of 1.0 nM GAPDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56°C).     

Binding free energy calculations of galectin-3-ligand interactions

Galectins show remarkable binding specificity towards beta-galactosides. A recently developed method for calculating binding free energies between a protein and its substrates has been used to evaluate the binding specificity of galectin-3. Five disaccharides and a tetrasaccharide were used as the substrates. The calculated binding free energies agree quite well with the experimental data and the ranking of binding affinities is well reproduced. For all the six protein-ligand complexes it was observed that electrostatic interactions oppose binding whereas the non-polar contributions drive complex formation. The observed binding specificity of galectin-3 for galactosides rather than glucosides is discussed in light of our results.     

Reversal of slow flow phenomenon during primary stenting by bail-out administration of abciximab

BACKGROUND: Slow flow or no reflow phenomenon is increasingly being recognized as a serious problem during coronary angioplasty and stenting. This phenomenon is seen more often during angioplasty in highly thrombogenic milieux, especially in a setting of acute myocardial infarction. The treatment of this complication is often not satisfactory. In this study the authors assessed the efficacy of abciximab, a potent antiplatelet drug, in treating slow flow or no reflow phenomenon during primary percutaneous transluminal coronary angioplasty (PTCA) for acute myocardial infarction (AMI). METHODS: Twenty-one instances of persistent slow flow phenomenon were encountered in 131 consecutive patients subjected to primary PTCA for AMI (16%). It was more common in patients presenting with AMI complicated by cardiogenic shock (nine of 21, 43%). Of these 21 cases of slow flow, 10 patients were given injection abciximab during the procedure of primary PTCA as a bail-out measure after encountering the complication of slow flow or no reflow. A pre-discharge coronary angiography was carried out in all patients who survived. RESULTS: In seven of 10 patients in the abciximab group flow had improved to TIMI-3. In contrast, in the non-abciximab group TIMI flow improved in only four of 11 patients. Patients with persistent slow flow had significantly higher mortality at the first 30-day follow-up than patients with TIMI-3 flow (33% versus 1.8%, p<0.001). CONCLUSION: In this small nonrandomized study significant improvement in coronary flow was achieved by using intravenous abciximab after observing slow flow or no reflow phenomenon during primary PTCA. More frequent use of this drug in this milieu might help in preventing the development of this complication. Larger studies are warranted to confirm this life-saving beneficial effect of bail-out administration of abciximab during primary angioplasty.