Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting

Plant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY‐2 cells expressing the human antibody M12 by selecting the co‐expressed fluorescent marker protein DsRed located on the same T‐DNA. Separation yielded ～35% wells containing single protoplasts and ～15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90–96% in the six monoclonal lines resulted in an up to 13‐fold increase in M12 production that remained stable for 10–12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.     

Synthesis and SAR-study for novel arylpiperazine derivatives of 5-arylidenehydantoin with α₁-adrenoceptor antagonistic properties

The study is focused on a series of 5-arylidenehydantoin derivatives with a phenylpiperazine-hydroxypropyl fragment at N3 of the hydantoin ring. The compounds were assessed on their affinity for α(1)-adrenoceptors and evaluated in functional bioassays for their antagonistic properties. Crystal structures of (Z)-5-(4-chlorobenzylidene)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)imidazolidine-2,4-dione (7) and hydrochloride of (Z)-5-(4-chlorobenzylidene)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (10a) were solved using the X-ray diffraction method. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) were used to predict 3D structure of compounds 5a-18a using a crystal structure of 7. SAR analysis was performed on the basis of Barbaro's pharmacophore model and structural properties of previously investigated α(1)-adrenoceptor antagonists possessing a hydantoin fragment. Most of the compounds exhibited significant affinities for α(1)-ARs in nanomolar range (40-290 nM). The highest activities (K(i)<75 nM) were observed for compounds possessing a 2-alkoxyphenylpiperazine fragment and two methoxy substituents at the benzylidene moiety. The results indicated that chemical properties, number and positions of substituents at the 5-arylidene fragment influenced the power of α(1)-affinities as follows: 3,4-di CH(3)O>2,4-di CH(3)O>4-Cl>2,3-di CH(3)O>H>4-N(CH(3))(2).     

Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.     

Combined surgery and radiotherapy in the treatment of ductal carcinoma in situ of the breast: preliminary results of the Hungarian multicenter prospective randomised study

The aim of this work is to report the preliminary results of the Hungarian multicentric randomised DCIS study. Between 2000 and 2007, 278 patients with ductal carcinoma in situ (DCIS) treated by breast-conserving surgery were randomised according to predetermined risk groups. Low/intermediate-risk patients (n=29) were randomised to 50 Gy whole-breast irradiation (WBI) or observation. High-risk cases (n=235) were allocated to receive 50 Gy WBI vs. 50 Gy WBI plus 16 Gy tumour bed boost. Very high-risk patients (patients with involved surgical margins; n=14) were randomised to 50 Gy WBI plus 16 Gy tumour bed boost or reoperation (reexcision plus radiotherapy or mastectomy alone). Immunohistochemistry (IHC) was performed to detect the expression of potential molecular prognostic markers (ER, PR, Her2, p53, Bcl-2 and Ki-67). At a median follow-up of 36 months no recurrence was observed in the low/intermediate- and very high-risk patient groups. In the high-risk group, 4 (1.7%) local recurrences and 1 (0.4%) distant metastasis occurred. No patient died of breast cancer. In the high-risk group of patients, the 3- and 5-year probability of local recurrence was 1.1% and 3.1%, respectively. The positive immunostaining for Her2 (38%), p53 (37%) and Ki-67 (44%) correlated with a high nuclear grade. Significant inverse correlation was found between the expression of ER (77%), PR (67%), Bcl-2 (64%) and grade. Preliminary results suggest that breast-conserving surgery followed by radiotherapy yields an annual local recurrence rate of less than 1% in patients with DCIS. IHC of molecular prognostic markers can assist to gain insight into the biologic heterogeneity of DCIS.     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Solution structure of the calponin homology (CH) domain from the smoothelin-like 1 protein: a unique apocalmodulin-binding mode and the possible role of the C-terminal type-2 CH-domain in smooth muscle relaxation

The SMTNL1 protein contains a single type-2 calponin homology (CH) domain at its C terminus that shares sequence identity with the smoothelin family of smooth muscle-specific proteins. In contrast to the smoothelins, SMTNL1 does not associate with F-actin in vitro, and its specific role in smooth muscle remains unclear. In addition, the biological function of the C-terminal CH-domains found in the smoothelin proteins is also poorly understood. In this work, we have therefore determined the solution structure of the CH-domain of mouse SMTNL1 (SMTNL1-CH; residues 346-459). The secondary structure and the overall fold for the C-terminal type-2 CH-domain is very similar to that of other CH-domains. However, two clusters of basic residues form a unique surface structure that is characteristic of SMTNL1-CH. Moreover, the protein has an extended C-terminal alpha-helix, which contains a calmodulin (CaM)-binding IQ-motif, that is also a distinct feature of the smoothelins. We have characterized the binding of apo-CaM to SMTNL1-CH through its IQ-motif by isothermal titration calorimetry and NMR chemical shift perturbation studies. In addition, we have used the HADDOCK protein-protein docking approach to construct a model for the complex of apo-CaM and SMTNL1-CH. The model revealed a close interaction of SMTNL1-CH with the two Ca(2+) binding loop regions of the C-terminal domain of apo-CaM; this mode of apo-CaM binding is distinct from previously reported interactions of apo-CaM with IQ-motifs. Finally, we comment on the putative role of the CH-domain in the biological function of SMTNL1.     

Transport of beads by several kinesin motors

The movements of beads pulled by several kinesin-1 (conventional kinesin) motors are studied both theoretically and experimentally. While the velocity is approximately independent of the number of motors pulling the beads, the walking distance or run-length is strongly increased when more motors are involved. Run-length distributions are measured for a wide range of motor concentrations and matched to theoretically calculated distributions using only two global fit parameters. In this way, the maximal number of motors pulling the beads is estimated to vary between two and seven motors for total kinesin concentrations between 0.1 and 2.5 μg/ml or between 0.27 and 6.7 nM. In the same concentration regime, the average number of pulling motors is found to lie between 1.1 and 3.2 motors.