Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate

Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.     

Growth hormone therapy in boy with panhypopituitarism may induce pilomatricoma recurrence: case report

Pilomatricoma is usually a solitary subcutaneous nodule. Recurrence of the nodule after surgical excision is very rare. Pilomatricoma occurrence in patients with growth hormone (GH) deficiency has not been reported, yet. We report a 14-year-old boy with pilomatricoma and panhypopituitarism. After GH therapy had been started, we observed two relapses of previously completely excised pilomatricoma in the same location and a new pilomatricoma formation on the chin.     

Distinct patterns of MMP-9 and MMP-2 activity in slow and fast twitch skeletal muscle regeneration in vivo

Skeletal muscles exhibit great plasticity and an ability to reconstruct in response to injury. However, the repair process is often inefficient and hindered by the development of fibrosis. We explored the possibility that during muscle repair, the different regeneration ability of the fast (extensor digitorum longus; EDL) and slow twitch (Soleus) muscles depends on the differential expression of metalloproteinases (MMP-9 and MMP-2) involved in the remodeling of the extracellular matrix. Our results show that MMP-9 and MMP-2 are present in the intact muscle and are up-regulated after crush-induced muscle injury. The expression and the activity of these two enzymes depend on the type of muscle and the phase of muscle regeneration. In the regenerating Soleus muscle, elevated levels of MMP-9 occurred during the myolysis and reconstruction phase. In contrast, regenerating EDL muscles exhibited decreased MMP-9 levels during myolysis and increased MMP-2 activity at the reconstruction phase. Moreover, satellite cells (mononuclear myoblasts) derived from Soleus and EDL muscles showed no differences in localization or activity of MMP-9 and MMP-2 during proliferation and differentiation in vitro. MMP-9 activity was present during all stages of myoblast differentiation, whereas MMP-2 activity reached its highest level during myoblast fusion. We conclude that MMPs are involved in muscle repair, and that fast and slow twitch muscles exhibit different patterns of MMP-9 and MMP-2 activity.     

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.     

The reduction of (ImH)[<Emphasis Type="Italic">trans</Emphasis>-Ru<Superscript>III</Superscript>Cl<Subscript>4</Subscript>(dmso)(Im)] under physiological conditions: preferential reaction of the reduced complex with human serum albumin

A systematic study of the reduction of (ImH)[trans-RuCl(4)(dmso)(Im)] (NAMI-A; dmso is dimethyl sulfoxide, Im is imidazole), a promising antimetastasing agent, by L: -ascorbic acid under physiological conditions is reported. Under blood plasma conditions (pH 7.4, 0.1-0.15 M NaCl , 37 degrees C) the rapid reduction of trans-[Ru(III)Cl(4)(dmso)(Im)](-) results in the formation of trans-[Ru(II)Cl(4)(dmso)(Im)](2-) within seconds, and is followed by successive dissociation of the chloride ligands, whereas neither dmso nor imidazole ligands are released during the reaction. Under our experimental conditions, the formation of the ascorbate dianion is the rate-determining step, and once it has formed it reacts rapidly with NAMI-A. Moreover, the NAMI-A complex is very unstable at physiological pH (7.4); therefore, the hydrolysis of NAMI-A cannot be excluded as a competing reaction. During hydrolysis, aquated derivatives via stepwise dissociation of chloride and dmso ligands are formed, and most of these species have a higher redox potential and are expected to be even more easily reduced by ascorbic acid. Thus, it is very likely that the reduced form of NAMI-A or the reduction products of its hydrolytic derivatives react with albumin. The reaction of reduced NAMI-A with human serum albumin leads to the formation of stable adducts, with a binding efficiency very similar to that of the parent complex, viz., 3.2 +/- 0.3 and 4.0 +/- 0.4 mol of Ru(II) and Ru(III) per mole of albumin, respectively, however with a significantly higher reactivity.     

Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.     

Spectroscopy and photophysics of flavin-related compounds: 3-benzyl-lumiflavin.

Molecular structure, spectroscopic and photophysical data for the singlet state of 3-benzyl-lumiflavin in different solvents are presented. Theoretical studies concerning singlet-singlet and triplet-triplet excitation energies were carried out using time-dependent density functional theory (TD-DFT) calculations. These predictions are in good agreement with the experimental results, which reflect the solvent interactions. All the observable singlet-singlet transitions have pi-pi* character. The title compound appears to be an efficient sensitizer of the production of singlet oxygen (phi(Delta)= 0.53). The crystal structure of 3-benzyl-lumiflavin is also presented, along with its solid-state photophysical data.     

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.     

Association analysis of vitamin D receptor gene polymorphisms with bone mineral density in young women with Graves' disease

Graves' (GD) hyperthyroidism induces accelerated bone turnover that leads to decreased bone mineral density (BMD). The role of the VDR gene in predisposition to primary osteoporosis has been recognized. Recent studies show associations between the VDR gene polymorphisms and susceptibility to autoimmune diseases. Here we analyzed if VDR gene polymorphisms: BsmI, ApaI, TaqI, and FokI may predispose women with Graves' hyperthyroidism to BMD reduction or to disease development. The subjects were 75 premenopausal female Polish patients with GD and 163 healthy women. The genotyping was performed by the use of the restriction fragment length polymorphism analysis (RFLP). We studied the association of the VDR polymorphisms and their haplotypes with patients' BMD and also SNPs and haplotypes association with Graves' disease. We found a strong linkage disequilibrium for the BsmI, ApaI, and TaqI polymorphims that formed three most frequent haplotypes in Graves' women: baT (47.9%), BAt (34.9%), and bAT (16.4%). We did not show statistically significant association of analyzed VDR polymorphisms or haplotypes with decreased bone mineral density in Graves' patients. However, the presence of F allele had a weak tendency to be associated with Graves' disease (with OR=1.93; 95% CI: 0.97-3.84; p=0.058). In conclusion: VDR gene polymorphisms do not predict the risk of decreased BMD in Polish women with Graves'. It may be speculated that the F allele carriers of the VDR-FokI polymorphism are predisposed to Graves' disease development.