Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.     

<Emphasis Type="Italic">Marcetia candolleana</Emphasis> (<Emphasis Type="Italic">Melastomeae</Emphasis> – <Emphasis Type="Italic">Melastomataceae</Emphasis>), a new species from Bahia (Brazil)

Summary   Marcetia candolleana A. K. A. Santos & A. B. Martins, is apparently restricted to Mucugê, Bahia (Brazil), where it occurs in areas of campo rupestre vegetation. This new species is closely related to the sympatric M. mucugensis Wurdack, but can be easily recognised by its semi-prostate to procumbent habit, reddish glandular-hirsute indument, loose and flexuous branches, leaves with inconspicuous reticulation on the abaxial surface, connectives very shortly prolonged below the thecae, style curved towards the apex, not exceeding the anthers, and pendulous fruit.

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Resumo   Marcetia candolleana A. K. A. Santos & A. B. Martins, é aparentemente restrita a Mucugê, Bahia (Brasil), onde ocorre em áreas de campo rupestre. Esta nova espécie é proximamente relacionada à M. mucugensis Wurdack, mas pode ser facilmente reconhecida por seu hábito semi-prostado a procumbente, indumento glandular-hirsuto, vináceo, ramos flexuosos, folhas inconspicuamente reticuladas na face abaxial, conectivos muito curtamente prolongados abaixo das tecas, estilete curvo no ápice, n?o ultrapassando o comprimento das anteras, e fruto pêndulo.

     

Synthesis and proteomic activity evaluation of a new isotope-coded affinity tagging (ICAT) reagent

During recent years, quantitative proteome profiling has taken advantage of incorporating the traditional stable isotope dilution analysis into global scale or discovery-based proteomic experiments that use mass spectrometers as detectors to allow the pairwise study of differently expressed proteins. Quantitative protein analysis by means of the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pairwise comparison of protein expression levels in biological samples. Herein, a modified ICAT reagent, named BAA-ICAT (beta-alanine-arm-ICAT) in which the polyether linker is replaced by a more water-soluble polyamide one, was investigated.     

Pichia angusta is an effective biocontrol yeast against postharvest decay of apple fruit caused by Botrytis cinerea and Monilia fructicola

The efficacy of eight isolates of Pichia angusta against three common postharvest pathogens of apple fruit was evaluated for the first time. All tested strains showed significant biocontrol activity against both Botrytis cinerea and Monilia fructicola , whereas efficacy against Penicillium expansum was poor. A leucine-auxotrophic mutant had no significant biocontrol activity against brown rot of apple, while the addition of 0.6–1.2 g L⁻¹ leucine in the fruit wound fully restored the biocontrol activity of this mutant against M. fructicola . Given the extremely well-developed classical and molecular genetics, the availability of genomic libraries, and its complete genomic sequence, this species can serve to elucidate the mechanisms related to biocontrol capacity.     

Leakage-free rapid quenching technique for yeast metabolomics

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at −40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at ≤−40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary ¹³C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Proteomic analysis of protein S-nitrosylation

Nitric oxide (NO) produces covalent PTMs of specific cysteine residues, a process known as S-nitrosylation. This route is dynamically regulated and is one of the major NO signalling pathways known to have strong and dynamic interactions with redox signalling. In agreement with this scenario, binding of NO to key cysteine groups can be linked to a broad range of physiological and pathological cellular events, such as smooth muscle relaxation, neurotransmission and neurodegeneration. The characterization of S-nitrosylated residues and the functional relevance of this protein modification are both essential information needed to understand the action of NO in living organisms. In this review, we focus on recent advances in this field and on state-of-the-art proteomic approaches which are aimed at characterizing the S-nitrosylome in different biological backgrounds.     

Evaluation of dried distillers' grains with solubles (DDGS) as a protein source for broilers

Male broiler chicks (n=120) were fed diets containing 0, 5, 10 or 15% dried distillers' grains with solubles (DDGS) from the 12th day up to the end of fattening (day 35). During this period feed intake, weight gain and excreta quality (pH, DM) were tested. A digestibility trial was carried out on four birds from each group on the last five days of the experiment to determine the digestibility of organic matter and CP of the different diets. The protein digestibility was evaluated using three different methods; uric acid correction, alpha-amino-N and amino acid-N. There were no significant effects of increased DDGS levels on feed intake, weight gain, excreta quality or digestibility of CP and organic matter. However, feed conversion showed a tendency to decline at the highest DDGS level (15%). Digestibility of DDGS protein was estimated to be 77%. There was no significant difference between uric acid and alpha-amino-N method, but both methods had a significantly lower CP digestibility than amino acid-N. The present results indicate that DDGS can be used as a protein source in diets for fattening broilers up to 10-15%.     

Measuring variability in trophic status in the Lake Waco/Bosque River Watershed

Background

Nutrient management in rivers and streams is difficult due to the spatial and temporal variability of algal growth responses. The objectives of this project were to determine the spatial and seasonal in situ variability of trophic status in the Lake Waco/Bosque River watershed, determine the variability in the lotic ecosystem trophic status index (LETSI) at each site as indicators of the system's nutrient sensitivity, and determine if passive diffusion periphytometers could provide threshold algal responses to nutrient enrichment.

Methods

We used the passive diffusion periphytometer to measure in-situ nutrient limitation and trophic status at eight sites in five streams in the Lake Waco/Bosque River Watershed in north-central Texas from July 1997 through October 1998. The chlorophyll a production in the periphytometers was used as an indicator of baseline chlorophyll a productivity and of maximum primary productivity (MPP) in response to nutrient enrichment (nitrogen and phosphorus). We evaluated the lotic ecosystem trophic status index (LETSI) using the ratio of baseline primary productivity to MPP, and evaluated the trophic class of each site.

Results

The rivers and streams in the Lake Waco/Bosque River Watershed exhibited varying degrees of nutrient enrichment over the 18-month sampling period. The North Bosque River at the headwaters (NB-02) located below the Stephenville, Texas wastewater treatment outfall consistently exhibited the highest degree of water quality impact due to nutrient enrichment. Sites at the outlet of the watershed (NB-04 and NB-05) were the next most enriched sites. Trophic class varied for enriched sites over seasons.

Conclusion

Seasonality played a significant role in the trophic class and sensitivity of each site to nutrients. Managing rivers and streams for nutrients will require methods for measuring in situ responses and sensitivities to nutrient enrichment. Nutrient enrichment periphytometers show significant potential for use in nutrient gradient studies.