A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene.

A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.     

The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp¹ directs the synthesis of a COOH terminally truncated or internally deleted peptide of ~124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH₂-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34^cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.     

Hemagglutination properties of Enterococcus

In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with -d-fucose, -d-galactose, -d-galactose, d-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes.     

Evaluation of a rapid method for the quantitative estimation of coliforms in meat by impedimetric procedures.

A 24-h instrumental procedure is described for the quantitative estimation of coliforms in ground meat. The method is simple and rapid, and it requires but a single sample dilution and four replicates. The data are recorded automatically and can be used to estimate coliforms in the range of 100 to 10,000 organisms per g. The procedure is an impedance detection time (IDT) method using a new medium, tested against 131 stock cultures, that markedly enhances the impedance response of gram-negative organisms, and it is selective for coliforms. Seventy samples of ground beef were analyzed for coliforms by the IDT method and the conventional three-dilution, two-step most-probable-number test tube procedure. Seventy-nine percent of the impedimetric estimates fell within the 95% confidence limits of the most-probable-number values. This corresponds to the criteria used to evaluate other coliform tests, with the added advantage of a single dilution and more rapid results.     

Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin.

Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzes the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It is isoelectric near pH 5.2 and has a molecular weight of approximately 71 000. The enzyme also hydrolyzes the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg, and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but does not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzes the Pro7-Phe8 peptide bond in Bk. It is isoelectric at pH 4.9 and has a molecular weight of approximately 68 000. Brain kininase B also hydrolyzes the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg, and Phe-Ser-Pro-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. However, kininase A and B hydrolyze the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gin-Val. The data show that a large part of the C-terminal portion of bradykinin is important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissle bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.     

Sgt1, a co‐chaperone of Hsp90 stabilizes Polo and is required for centrosome organization

Sgt1 was described previously in yeast and humans to be a Hsp90 co‐chaperone and required for kinetochore assembly. We have identified a mutant allele of Sgt1 in Drosophila and characterized its function. Mutations in sgt1 do not affect overall kinetochore assembly or spindle assembly checkpoint. sgt1 mutant cells enter less frequently into mitosis and arrest in a prometaphase‐like state. Mutations in sgt1 severely compromise the organization and function of the mitotic apparatus. In these cells, centrioles replicate but centrosomes fail to mature, and pericentriolar material components do not localize normally resulting in highly abnormal spindles. Interestingly, a similar phenotype was described previously in Hsp90 mutant cells and correlated with a decrease in Polo protein levels. In sgt1 mutant neuroblasts, we also observe a decrease in overall levels of Polo. Overexpression of the kinase results in a substantial rescue of the centrosome defects; most cells form normal bipolar spindles and progress through mitosis normally. Taken together, these findings suggest that Sgt1 is involved in the stabilization of Polo allowing normal centrosome maturation, entry and progression though mitosis.     

JmjC-KDMs KDM3A and KDM6B modulate radioresistance under hypoxic conditions in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the most frequent esophageal cancer (EC) subtype, entails dismal prognosis. Hypoxia, a common feature of advanced ESCC, is involved in resistance to radiotherapy (RT). RT response in hypoxia might be modulated through epigenetic mechanisms, constituting novel targets to improve patient outcome. Post-translational methylation in histone can be partially modulated by histone lysine demethylases (KDMs), which specifically removes methyl groups in certain lysine residues. KDMs deregulation was associated with tumor aggressiveness and therapy failure. Thus, we sought to unveil the role of Jumonji C domain histone lysine demethylases (JmjC-KDMs) in ESCC radioresistance acquisition. The effectiveness of RT upon ESCC cells under hypoxic conditions was assessed by colony formation assay. KDM3A/KDM6B expression, and respective H3K9me2 and H3K27me3 target marks, were evaluated by RT-qPCR, Western blot, and immunofluorescence. Effect of JmjC-KDM inhibitor IOX1, as well as KDM3A knockdown, in in vitro functional cell behavior and RT response was assessed in ESCC under hypoxic conditions. In vivo effect of combined IOX1 and ionizing radiation treatment was evaluated in ESCC cells using CAM assay. KDM3A, KDM6B, HIF-1α, and CAIX immunoexpression was assessed in primary ESCC and normal esophagus. Herein, we found that hypoxia promoted ESCC radioresistance through increased KDM3A/KDM6B expression, enhancing cell survival and migration and decreasing DNA damage and apoptosis, in vitro. Exposure to IOX1 reverted these features, increasing ESCC radiosensitivity and decreasing ESCC microtumors size, in vivo. KDM3A was upregulated in ESCC tissues compared to the normal esophagus, associating and colocalizing with hypoxic markers (HIF-1α and CAIX). Therefore, KDM3A upregulation in ESCC cell lines and primary tumors associated with hypoxia, playing a critical role in EC aggressiveness and radioresistance. KDM3A targeting, concomitant with conventional RT, constitutes a promising strategy to improve ESCC patients’ survival.Subject terms: Predictive markers, Cancer     

Autophagy inhibition radiosensitizes in vitro,yet reduces radioresponses in vivo due to deficient immunogenic signalling

Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers.