Dual mode of degradation of Cdc25 A phosphatase

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment.     

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides

There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry. In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures. The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791). Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E. coli cells subjected to prior thermal permeabilisation. Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75°C). Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest. Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio. The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75°C. The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml⁻¹ h⁻¹ (g biocatalyst)⁻¹ with ~40% conversion when using a 30% (w/v) solution.     

Assessment of in-line near-infrared spectroscopy for continuous monitoring of fermentation processes

The application of NIR in-line to monitor and control fermentation processes was investigated. Determination of biomass, glucose, and lactic and acetic acids during fermentations of Staphylococcus xylosus ES13 was performed by an interactance fiber optic probe immersed into the culture broth and connected to a NIR instrument. Partial least squares regression (PLSR) calibration models of second derivative NIR spectra in the 700-1800 nm region gave satisfactory predictive models for all parameters of interest: biomass, glucose, and lactic and acetic acids. Batch, repeated batch, and continuous fermentations were monitored and automatically controlled by interfacing the NIR to the bioreactor control unit. The high frequency of data collection permitted an accurate study of the kinetics, supplying lots of data that describe the cultural broth composition and strengthen statistical analysis. Comparison of spectra collected throughout fermentation runs of S. xylosus ES13, Lactobacillus fermentum ES15, and Streptococcus thermophylus ES17 demonstrated the successful extension of a unique calibration model, developed for S. xylosus ES13, to other strains that were differently shaped but growing in the same medium and fermentation conditions. NIR in-line was so versatile as to measure several biochemical parameters of different bacteria by means of slightly adapted models, avoiding a separate calibration for each strain.     

Crystal structure of a zinc-activated variant of human carbonic anhydrase I,CA I Michigan 1: evidence for a second zinc binding site involving arginine coordination

The human genetic variant carbonic anhydrase I (CA I) Michigan 1 results from a single point mutation that changes His 67 to Arg in a critical region of the active site. This variant of the zinc metalloenzyme appears to be unique in that it possesses an esterase activity that is specifically enhanced by added free zinc ions. We have determined the three-dimensional structure of human CA I Michigan 1 by X-ray crystallography to a resolution of 2.6 A. In the absence of added zinc ions, the mutated residue, Arg 67, points out of the active site, hydrogen bonding with the carboxylate of Asn 69. This contrasts with the orientation of His 67, in the native isozyme, which points into the active site. The orientations of His 94, His 96, and His 119, that coordinate the catalytic zinc ion, and of the catalytically critical Thr 199-Glu 106 hydrogen bonding system, are largely unchanged in the mutant. The structure of an enzyme adduct with a second zinc bound was determined to a resolution of 2.0 A. The second zinc ion is coordinated to His 64, His 200, and Arg 67. This arginine residue reverses its orientation on zinc binding and turns into the active site. The residues at these three positions have been implicated in determining the specific kinetic properties of native CA I. This is, to our knowledge, the first example of a zinc ion coordinating with an arginine residue in a Zn(II) enzyme.     

A simple non invasive computerized method for the assessment of bone repair within osteoconductive porous bioceramic grafts

Single energy X-ray imaging, due to its low cost and flexibility, is one of the most used and common technique to assess bone state and bone remodeling over time. Standardized X-ray images are needed to compare sets of radiographs for semi-quantitative analyses of tissue remodeling. However, useful mathematical modeling for the analysis of high level radiographic images are not easily available. In order to propose a useful evaluation tool to a wide clinical scenario, we present an innovative calibration algorithm for a semi-quantitative analysis of non-standardized digitized X-ray images. For calibration on a unique standardization scale, three time invariant regions (ROI) of radiographs were selected and analyzed. The accuracy of the normalization method for X-ray films was successfully validated by using an aluminum step wedge for routine X-ray exposures as tool to standardize serial radiographs (Pearson correlation test: R(2) = 0.96). This method was applied to investigate the progression of the new bone deposition within ceramic scaffolds used as osteoconductive substitute in large bone defects taking advantage of a large animal model. This innovative image-processing algorithm allowed the identification and semi-quantification of the bone matrix deposited within the implant. The osteo-integration at the bone-implant interface was also investigated. A progressively increasing bone tissue deposition within the porous bioceramic implant and a progressive osteo-integration was observed during the 12 months of the trial.     

Periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for BK virus nephropathy in renal transplant patients

OBJECTIVE: To investigate the significance of polyomavirus (PV) viruria and viremia by morphologic, immunohistochemical and molecular analysis (multiplex nested-polymerase chain reaction) in renal transplant patients. STUDY DESIGN: Urine (n=328), serum (n= 53) and renal biopsies (n=24) from renal transplant patients (n=106) were studied. RESULTS: Decoy cells were found in 53 samples (16%) from 19 patients (18%); viral DNA was amplified in all urinary samples and disclosed BK virus (BKV) (n=24), JC virus (JCV) (n=16), and JCV and BKV DNA (n=13). BKV was the prevailing genotype in patients with a high frequency of decoy cell excretion (p = 0.001). JCV excretion correlated with a low number (p = 0.01) and BKV with a high number of decoy cells (p=0.003). PV DNA was amplified from 30/53 serum samples (56.6%); BKV was the prevailing genotype (p = 0.04). On 24 renal biopsies (18 from the decoy cell-negative and 6 from the decoy cell-positive group) PV nephropathy (PVN) was identified and BKV DNA amplified in 4 biopsies, all from the group with a high frequency of decoy cell excretion. PVN was not identified in renal biopsies from the decoy cell-negative group. CONCLUSION: PV infection is frequent in renal transplant patients. The BKV genotype in urine and serum is significantly related to a high frequency and high number of decoy cells. PVN occurs only in patients with BKV viremia and a high number and frequency of decoy cell excretion in urine. In the absence of decoy cells, PVN can be excluded. Cytologic analysis of urine is an important diagnostic tool for screening renal transplant patients at risk of PVN.     

Calmodulin-dependent protein kinase IV regulates hematopoietic stem cell maintenance

The hematopoietic stem cell (HSC) gives rise to all mature, terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure, characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn, these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2.     

The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module

The possibility of predicting the overall shape of a macromolecule in solution from its diffusional properties has gained increasing importance in the structural genomic era. Here we explore and quantify the influence that unstructured and flexible regions have on the motions of a globular protein, a situation that can occur from the presence of such regions in the natural sequence or from additional tags. I27, an immunoglobulin-like module from the muscle protein titin, whose structure and properties are well characterized, was selected for our studies. The backbone dynamics and the overall tumbling of three different constructs of I27 were investigated using (15)N NMR relaxation collected at two (15)N frequencies (60.8 and 81.1 MHz) and fluorescence depolarization spectroscopy after labeling of a reactive cysteine with an extrinsic fluorophore. Our data show that the presence of disordered tags clearly exerts a frictional drag that increases with the length of the tags, thus affecting the module tumbling in solution. We discuss the use and the limitations of current approaches to hydrodynamic calculations, especially when having to take into account local flexibility.     

A proteomics strategy for the enrichment of receptor-associated complexes

Multimeric protein complexes are important for cell function and are being identified by proteomics approaches. Enrichment strategies, such as those employing affinity matrices, are required for the characterization of such complexes, for example, those containing growth factor receptors. The receptor for the macrophage lineage growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1), is the tyrosine kinase, c-Fms. There is evidence that the CSF-1 receptor (CSF-1R) forms distinct multimeric complexes involving autophosphorylated tyrosines in its cytoplasmic region; however, these complexes are difficult to identify by immunoprecipitation, making enrichment necessary. We report here the use of a tyrosine-phosphorylated, GST-fusion construct of the entire CSF-1R cytoplasmic region to characterize proteins putatively associating with the activated CSF-1R. Besides signalling molecules known to associate with the receptor or be involved in CSF-1R-dependent signalling, mass spectrometry identified a number of other molecules binding to the construct. So far among these candidate proteins, dynein, claudin and silencer of death domains co-immunoprecipitated with the CSF-1R, suggesting association. This affinity matrix method, using an entire cytoplasmic region, may have relevance for other growth factor receptors.