Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination

   

Recently Mycobacterium tuberculosis was shown to possess a novel protein modification, in which a small protein Pup is conjugated to the epsilon-amino groups of lysines in target proteins. Analogous to ubiquitin modification in eukaryotes, this remarkable modification recruits proteins for degradation via archaeal-type proteasomes found in mycobacteria and allied actinobacteria. While a mycobacterial protein named PafA was found to be required for this conjugation reaction, its biochemical mechanism has not been elucidated. Using sensitive sequence profile comparison methods we establish that the PafA family proteins are related to the γ-glutamyl-cysteine synthetase and glutamine synthetase. Hence, we predict that PafA is the Pup ligase, which catalyzes the ATP-dependent ligation of the terminal γ-carboxylate of glutamate to lysines, similar to the above enzymes. We further discovered that an ortholog of the eukaryotic PAC2 (e.g. cg2106) is often present in the vicinity of the actinobacterial Pup-proteasome gene neighborhoods and is likely to represent the ancestral proteasomal chaperone. Pup-conjugation is sporadically present outside the actinobacteria in certain lineages, such as verrucomicrobia, nitrospirae, deltaproteobacteria and planctomycetes, and in the latter two lineages it might modify membrane proteins.     

Normalizing and scaling of data to derive human response corridors from impact tests

It is well known that variability is inherent in any biological experiment. Human cadavers (Post-Mortem Human Subjects, PMHS) are routinely used to determine responses to impact loading for crashworthiness applications including civilian (motor vehicle) and military environments. It is important to transform measured variables from PMHS tests (accelerations, forces and deflections) to a standard or reference population, termed normalization. The transformation process should account for inter-specimen variations with some underlying assumptions used during normalization. Scaling is a process by which normalized responses are converted from one standard to another (example, mid-size adult male to large-male and small-size female adults, and to pediatric populations). These responses are used to derive corridors to assess the biofidelity of anthropomorphic test devices (crash dummies) used to predict injury in impact environments and design injury mitigating devices. This survey examines the pros and cons of different approaches for obtaining normalized and scaled responses and corridors used in biomechanical studies for over four decades. Specifically, the equal-stress equal-velocity and impulse-momentum methods along with their variations are discussed in this review. Methods ranging from subjective to quasi-static loading to different approaches are discussed for deriving temporal mean and plus minus one standard deviation human corridors of time-varying fundamental responses and cross variables (e.g., force-deflection). The survey offers some insights into the potential efficacy of these approaches with examples from recent impact tests and concludes with recommendations for future studies. The importance of considering various parameters during the experimental design of human impact tests is stressed.     

The optimal flow rate and column length for maximum production rate of protein A affinity chromatography

Production rate is an important parameter in the design of efficient protein A affinity chromatography processes for purifying recombinant monoclonal antibodies. A simple equation was derived that expresses production rate in terms of flow rate and column length. Changes in flow rate and column length will not affect the antibody and are therefore easily varied for bioprocess applications. In the equation, production rate depends on dynamic capacity, which can be expressed as a function of the load flow rate and column length. The only empirical data needed for production rate optimization is the relationship of dynamic capacity to load flow rate and column length, which was quickly determined by using an on-line assay. The optimal production rate was found at a high flow rate, a low column length, and a low dynamic capacity, which has several implications for using high production rate protein A affinity chromatography for antibody manufacturing.     

KIBRA Regulates Aurora Kinase Activity and Is Required for Precise Chromosome Alignment During Mitosis

The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser⁸³ by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser⁵³⁹, probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.     

Changes in Vegetation Characteristics Downwind of an Aluminium Factory in India

Vegetation characteristics were assessed at different distancesdownwind of an aluminium factory to elucidate changes in communityattributes. The importance value index (IVI) of sensitive speciesdecreased and those of tolerant species increased with the increasingpollution load around the factory. Plant community compositionchanged with distance from this source. Species richness andthe Shannon-Weiner index increased with the increasing distancefrom the source, while concentration of dominance declined from1 to 11 km. The similarity coefficient indicated that the thirdand fifth sites are closer to each other, and first and fourthsites are farthest. The shape of the species sequence vs. theIVI distribution curve slipped from the shape of log normaldistribution at sites receiving higher pollution load. The woodylayer is found to be more affected compared to the herbaceouslayer. The aluminium factory impact was most pronounced up to4 km downwind of the source. Data on ambient air quality werefound to be directly related to the unfavourable changes incommunity attributes.Copyright 1994, 1999 Academic Press Importance value index, aluminium factory, community attributes, herbaceous layer, woody layer, sensitive and tolerant species