Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability

Accurate control of the number of centrosomes, the major microtubule-organizing centers of animal cells, is critical for the maintenance of genome integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation, and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material, and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication, and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Using chemical genetics, we show that kinase-active Plk4 is inherently unstable and targeted for degradation. Plk4 is shown to multiply self-phosphorylate within a 24–amino acid phosphodegron. Phosphorylation of multiple sites is required for Plk4 instability, indicating a requirement for a threshold level of Plk4 kinase activity to promote its own destruction. We propose that kinase-mediated, autoregulated instability of Plk4 self-limits Plk4 activity so as to prevent centrosome amplification.     

Two alanine aminotranferases link mitochondrial glycolate oxidation to the major photorespiratory pathway in Arabidopsis and rice

The major photorespiratory pathway in higher plants is distributed over chloroplasts, mitochondria, and peroxisomes. In this pathway, glycolate oxidation takes place in peroxisomes. It was previously suggested that a mitochondrial glycolate dehydrogenase (GlcDH) that was conserved from green algae lacking leaf-type peroxisomes contributes to photorespiration in Arabidopsis thaliana. Here, the identification of two Arabidopsis mitochondrial alanine:glyoxylate aminotransferases (ALAATs) that link glycolate oxidation to glycine formation are described. By this reaction, the mitochondrial side pathway produces glycine from glyoxylate that can be used in the glycine decarboxylase (GCD) reaction of the major pathway. RNA interference (RNAi) suppression of mitochondrial ALAAT did not result in major changes in metabolite pools under standard conditions or enhanced photorespiratroy flux, respectively. However, RNAi lines showed reduced photorespiratory CO(2) release and a lower CO(2) compensation point. Mitochondria isolated from RNAi lines are incapable of converting glycolate to CO(2), whereas simultaneous overexpression of GlcDH and ALAATs in transiently transformed tobacco leaves enhances glycolate conversion. Furthermore, analyses of rice mitochondria suggest that the side pathway for glycolate oxidation and glycine formation is conserved in monocotyledoneous plants. It is concluded that the photorespiratory pathway from green algae has been functionally conserved in higher plants.     

Resin-based investigation of acyl carrier protein interaction networks in Escherichia coli

Protein-protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target.     

Quantitative analysis of gap-junctional intercellular communication in precision-cut mouse liver slices

Direct intercellular communication through gap junction channels is involved in the maintenance of tissue homeostasis and suppression of carcinogenesis. Gap-junctional communication is often altered in tumor cells but it can also be modulated in response to tumor promotors or inflammatory signals. In order to evaluate the effect of nongenotoxic compounds, suggested to be involved in tumor promotion, on gap junctional intercellular communication in the liver, we have developed a direct dye transfer method. The fluorescent dye Alexa Fluor 488 was iontophoretically injected into hepatocytes of freshly prepared, precision-cut mouse liver slices (250 microm). The area of dye spreading was monitored and quantified by microscopy. Comparison of dye spreading in connexin-32-deficient versus wild-type liver revealed a 96% decrease in connexin-32-deficient tissue. Induction of an acute phase response in connexin-32-deficient mice by intraperitoneal injection of lipopolysaccharide increased dye coupling by 33%, probably due to upregulation of connexin-26-containing gap junction channels.     

Deoxynivalenol in commercial beer — screening for the toxin with an indirect competitive ELISA

Antibodies specific for deoxynivalenol (DON) were prepared by immunizing rabbits with a deoxynivalenol-hemiglutaryl-HSA conjugate. The antibody showed cross-reactivity with DON, 15a-acetyl-DON (15-Ac DON), and 3a-acotyl-DON (3-Ac DON) of 100%, 216%, and 260%, respectively. No cross-reactivity was observed against 20 further trichothecenes of the A-, B-, and C-type. An indirect competitive ELISA procedure was set up for the detection of DON in liquid matrices occurring in the brewing process, Including beer. ELISA was sensitive down to 3.8 ng/mL. Detection limit for DON in tenfold diluted beer was 50 ppb. Recovery of the toxin in spiked beer was 45%, 77%, and 83% at toxin levels of 50; 500; and 1,000 ppb, respectively. ELISA results corresponded quite well with HPLC data. 196 commercial beers from several German breweries, including 36 gushing-positive samples, were assayed for DON. Screening revealed toxin levels from not detectable to 569 ppb. 69% of the gushing beers had marked levels of DON. 37% of the gushing-negative beer samples contained DON in detectable amounts with a maximum content of 172 ppb. Wheat beers had significantly higher DON-contents than beers derived from barley. Concentration of the toxin was significantly higher in gushing beers than in nongushing beers from both cereal sources.     

A high‐throughput sample preparation method for cellular proteomics using 96‐well filter plates

A high‐throughput sample preparation protocol based on the use of 96‐well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96‐well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel‐filtration columns.     

Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors

The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.     

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.