Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling.

Across eukaryotes, disruption of DNA replication causes an S phase checkpoint response, which regulates multiple processes, including inhibition of replication initiation and fork stabilization. How these events are coordinated remains poorly understood. Here, we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to forkhead-associated (FHA) interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally, we show that a Cdc45 mutation found in patients with Meier-Gorlin syndrome disrupts the functional interaction with Rad53 in yeast. Together, we present a single mechanism by which a checkpoint kinase targets replication initiation and elongation complexes, which may be relevant to human disease.

Human RecQL4 helicase plays multifaceted roles in the genomic stability of normal and cancer cells.

Human RecQ helicases that share homology with E. coli RecQ helicase play critical roles in diverse biological activities such as DNA replication, transcription, recombination and repair. Mutations in three of the five human RecQ helicases (RecQ1, WRN, BLM, RecQL4 and RecQ5) result in autosomal recessive syndromes characterized by accelerated aging symptoms and cancer incidence. Mutational inactivation of Werner (WRN) and Bloom (BLM) genes results in Werner syndrome (WS) and Bloom syndrome (BS) respectively. However, mutations in RecQL4 result in three human disorders: (I) Rothmund-Thomson syndrome (RTS), (II) RAPADILINO and (III) Baller-Gerold syndrome (BGS). Cells from WS, BS and RTS are characterized by a unique chromosomal anomaly indicating that each of the RecQ helicases performs specialized function(s) in a non-redundant manner. Elucidating the biological functions of RecQ helicases will enable us to understand not only the aging process but also to determine the cause for age-associated human diseases. Recent biochemical and molecular studies have given new insights into the multifaceted roles of RecQL4 that range from genomic stability to carcinogenesis and beyond. This review summarizes some of the existing and emerging knowledge on diverse biological functions of RecQL4 and its significance as a potential molecular target for cancer therapy.

Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication.

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

The KAT6B-related disorders genitopatellar syndrome and Ohdo/SBBYS syndrome have distinct clinical features reflecting distinct molecular mechanisms.

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) have both recently been shown to be caused by distinct mutations in the histone acetyltransferase KAT6B (a.k.a. MYST4/MORF). All variants are de novo dominant mutations that lead to protein truncation. Mutations leading to GPS occur in the proximal portion of the last exon and lead to the expression of a protein without a C-terminal domain. Mutations leading to SBBYSS occur either throughout the gene, leading to nonsense-mediated decay, or more distally in the last exon. Features present only in GPS are contractures, anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, and agenesis of the corpus callosum. Features present only in SBBYSS include long thumbs and long great toes and lacrimal duct abnormalities. Several features occur in both, such as intellectual disability, congenital heart defects, and genital and patellar anomalies. We propose that haploinsufficiency or loss of a function mediated by the C-terminal domain causes the common features, whereas gain-of-function activities would explain the features unique to GPS. Further molecular studies and the compilation of mutations in a database for genotype-phenotype correlations (www.LOVD.nl/KAT6B) might help tease out answers to these questions and understand the developmental programs dysregulated by the different truncations.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential.

BACKGROUND: Like all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span. METHODS: We transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo. RESULTS: Our results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components. CONCLUSIONS: Focal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry.

The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with ß-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC(®) BEH C18 column (10 cm L×2.1 mm ID with 1.7 µm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13 min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors' laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples.

How to remain nonfolded and pliable: the linkers in modular α-amylases as a case study.

The primary structure of linkers in a new class of modular α-amylases constitutes a paradigm of the structural basis that allows a polypeptide to remain nonfolded, extended and pliable. Unfolding is mediated through a depletion of hydrophobic residues and an enrichment of hydrophilic residues, amongst which Ser and Thr are over-represented. An extended and flexible conformation is promoted by the sequential arrangement of Pro and Gly, which are the most abundant residues in these linkers. This is complemented by charge repulsion, charge clustering and disulfide-bridged loops. Molecular dynamics simulations suggest the existence of conformational transitions resulting from a transient and localized hydrophobic collapse, arising from the peculiar composition of the linkers. Accordingly, these linkers should not be regarded as fully disordered, but rather as possessing various discrete structural patterns allowing them to fulfill their biological function as a free energy reservoir for concerted motions between structured domains.

Skeletal unloading induces a full-thickness patellar cartilage defect with increase of urinary collagen II CTx degradation marker in growing rats.

Mechanical stress plays an important role in tissue morphogenesis and extracellular matrix metabolism. However, little is known about the effects of reduced loading without restriction of joint motion on the patella. We investigated the effects of long-term skeletal unloading on patellar cartilage and subchondral bone and systemic collagen II metabolism. Nine-week-old male F344/N rats (n=128) were randomly divided into two groups: caged control (C) and tail suspended (TS). Hindlimbs of the TS rats were subjected to unloading for up to 12 weeks. Sequential changes in the patellar cartilage and subchondral bone were analyzed macroscopically, by pathological findings and histomorphologically. All animals received double tidemark fluorochrome labeling prior to sacrifice. Glycosaminoglycan (GAG) content in patellar cartilage, cross-linked C-telopeptide of type II collagen (CTx-II) in 24-h urine and type II procollagen-C-peptide (pCol-II-C) in sera were also measured by DMB assay, ELISA and EIA, respectively. In the TS group, GAG content was significantly reduced only during the first 3 weeks. No further significant decrease was found. Alkaline phosphatase (ALP) activity was increased, especially at the deep zone. Tidemark mineral apposition rate (MAR) was temporally increased, which resulted in an increase in the ratio of calcified cartilage to the entire cartilage. In the medial part, in addition, thickness of the entire cartilage was decreased by temporal acceleration of subchondral ossification advancement and, in the medial margin, a full-thickness cartilage defect was revealed in 88.6% of TS rats. However, the remaining articular surface was free from fibrillation. While urinary CTx-II was significantly increased during the experimental periods, serum pCol-II-C was significantly decreased at the early phase. There were significant correlations between the urinary CTx-II levels and tidemark MAR. Our results provided evidence that skeletal unloading increased ALP activity at the deep zone and temporally accelerated tidemark advancement associated with a decrease in proteoglycan content. In addition, skeletal unloading temporally accelerated subchondral ossification advancement in the medial part of the patella and finally induced a full-thickness patellar cartilage defect without any fibrillation at the remaining articular surface by additional subchondral bone modeling and possible retarded cartilage growth, which was through a different mechanism than overloading.

Stress deprivation enhances manganese superoxide dismutase expression in the rat patellar tendon.

Manganese superoxide dismutase (Mn-SOD) is a key antioxidant enzyme that prevents reactive oxygen species (ROS) damage to biological tissues. Although Mn-SOD has been investigated for a variety of cells, little is known about its expression in the tendon, particularly in the stress-deprived condition. The present study demonstrated that Mn-SOD is excessively expressed in the cultured fibroblasts derived from the stress-deprived patellar tendon in the rat using subtractive hybridization analysis. In addition, we confirmed that the expression of Mn-SOD is up-regulated in the stress-deprived patellar tendon in vivo at both the mRNA and protein levels. These results suggest that Mn-SOD may play a role in regulating ROS and matrix degradation involving mechanical deterioration of the stress-deprived tendon tissue.

Hydrolysis of conjugated metabolites of buprenorphine. I. The quantitative enzymatic hydrolysis of buprenorphine-3-beta-D-glucuronide in human urine.

Buprenorphine, which is a powerful analgesic, a substitution drug for opioids widely used in Europe, and a promising new drug currently undergoing clinical trials in the treatment of opioid dependence in the U.S., is excreted in human urine mainly as glucuronide conjugates. In gas chromatographic-mass spectrometric analysis, the urine specimens must be first hydrolyzed to release buprenorphine from its glucuronide conjugates. In order to evaluate the existing hydrolysis methods and to find the optimal hydrolysis conditions, buprenorphine-3-beta-D-glucuronide (B3G) was synthesized. Urine fortified with synthetic B3G was hydrolyzed using acid, base, and beta-glucuronidases from different source species, including Helix pomatia, Escherichia coli, and Patella vulgata. Glusulase, a preparation containing both beta-glucuronidase (H. pomatia) and sulfatase, was also tested. Whereas both acidic and basic hydrolysis were ineffective, quantitative hydrolysis could be achieved by using beta-glucuronidases under appropriate conditions. However, we found that there was a marked difference in the reactivity of these enzymes (E. coli > H. pomatia >> P. vulgata). The optimal incubation conditions for enzymatic hydrolysis of B3G were 2 h at 37 degrees C for E coli and 4 h at 60 degrees C or 16 h at 37 degrees C for H. pomatia. Using 1000 Fishman units of either of these two enzymes, effective hydrolysis could be achieved even when the B3G concentration was as high as 2000 ng/mL. Glusulase was equally effective toward B3G if the fortified urine samples were incubated with 25 microL of this enzyme for 1 h at 60 degrees C.

Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis.

The aim of this study was to characterize the cellular phenotypes of articular cartilage and meniscus in rabbits with experimentally induced osteoarthritis (OA), by histological and molecular biological techniques. OA was induced by severing the anterior cruciate ligament of the knee and rabbits were killed 2, 4 or 9 weeks following surgery. Our histological observations show a progressive destruction of extracellular matrix in both tissues. To determine whether these morphological changes could be related to alterations in the regulation of gene expression for a subset of relevant molecules, levels of mRNA for proteinases and one inhibitor (MMP-1, -3 and -13, aggrecanase-1 and -2 and TIMP-1), matrix molecules and one chaperone (type II and X collagens, aggrecan, osteonectin, betaig-h3 and BiP) were assessed by reverse transcription-polymerase chain reaction. Our results indicate that for most markers expression profiles were similar in both tissues. In particular, matrix protein gene expression remained stable or varied little during progression of OA, suggesting a poor repair capacity of the tissues. MMP gene expression increased rapidly whereas aggrecanase gene expression remained stable. These findings suggest that differential regulation of mRNA levels of MMP-1, -3 and -13 on the one hand and aggrecanase-1 and -2 on the other, occurs during OA.

Two latent metalloproteases of human articular cartilage that digest proteoglycan.

Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.

Neutral proteases and cathepsin D in human articular cartilage.

Proteolytic enzymes have been studied in extracts of human articular cartilage by the use of micromethods. The digestion of hemoglobin at pH 3.2 and of cartilage proteoglycan at pH 5 was shown to be due chiefly to cathepsin D. Cathepsin D was purified 900-fold from human patellar cartilage. Its identity was established by its specific cleavage of the B chain of insulin. At least six multiple forms of cathepsin D are present in cartilage; these corresponded to bovine forms 4-9. Cathepsin D had no action on proteins at pH 7.4. However, cartilage extracts digested proteoglycan, casein, and histone at this pH. The proteolytic activities against these three substrates were purified about 170-, 160-, and 70-fold, respectively. Each activity appeared in multiple forms on DEAE-Sephadex chromatography. The three activities appear to be different since cysteine inhibited casein digestion, aurothiomalate inhibited histone digestion, and neither inhibited proteoglycan digestion. Tests with a wide range of inhibitors and activators suggest that these three activities differ from other neutral proteases described in the literature.