The RAS Effector RIN1 Directly Competes with RAF and Is Regulated by 14-3-3 Proteins

Activation of RAS proteins can lead to multiple outcomes by virtue of regulated signal traffic through alternate effector pathways. We demonstrate that the RAS effector protein RIN1 binds to activated RAS with an affinity (K(d), 22 nM) similar to that observed for RAF1. At concentrations close to their equilibrium dissociation constant values, RIN1 and RAF1 compete directly for RAS binding. RIN1 was also observed to inhibit cellular transformation by activated mutant RAS. This distinguishes RIN1 from other RAS effectors, which are transformation enhancing. Blockade of transformation was mediated by the RAS binding domain but required membrane localization. RIN1 recognizes endogenous RAS following transient activation by epidermal growth factor, and a portion of RIN1 fractionates to the cell membrane in a manner consistent with a reversible interaction. RIN1 also binds to 14-3-3 proteins through a sequence including serine 351. Mutation of this residue abolished the 14-3-3 binding capacity of RIN1 and led to more efficient blockade of RAS-mediated transformation. The mutant protein, RIN1(S351A), showed a shift in localization to the plasma membrane. Serine 351 is a substrate for protein kinase D (PKD [also known as PKCmu]) in vitro and in vivo. These data suggest that the normal localization and function of RIN1, as well as its ability to compete with RAF, are regulated in part by 14-3-3 binding, which in turn is controlled by PKD phosphorylation.     

Effect of initial substrate concentration on the rate of nitrification in a batch experiment

The intrinsic rate of nitrification was observed in a batch reactor by eliminating external and internal diffusional resistances. The former were minimized by means of intense agitation, and the latter by mechanical rupture of the activated sludge flocs using high mixer rotational speeds. The optimum temperature and pH for the intrinsic nitrification rate were found to be 30–35°C and 8.0, respectively. Initial ammonium concentration was found to have a strong effect on the value of the kinetic parameters of the Michaelis–Menten rate expression at low ammonium levels. However, at high initial concentrations both parameters attained a constant maximum value that is independent of the initial substrate level.     

A weak link in metabolism: the metabolic capacity for glycine biosynthesis does not satisfy the need for collagen synthesis

In a previous paper, we pointed out that the capability to synthesize glycine from serine is constrained by the stoichiometry of the glycine hydroxymethyltransferase reaction, which limits the amount of glycine produced to be no more than equimolar with the amount of C 1 units produced. This constraint predicts a shortage of available glycine if there are no adequate compensating processes. Here, we test this prediction by comparing all reported fluxes for the production and consumption of glycine in a human adult. Detailed assessment of all possible sources of glycine shows that synthesis from serine accounts for more than 85% of the total, and that the amount of glycine available from synthesis, about 3 g/day, together with that available from the diet, in the range 1.5–3.0 g/day, may fall significantly short of the amount needed for all metabolic uses, including collagen synthesis by about 10 g per day for a 70 kg human. This result supports earlier suggestions in the literature that glycine is a semi-essential amino acid and that it should be taken as a nutritional supplement to guarantee a healthy metabolism.     

Correlation of Hypertension and Proteinuria with Outcome in Elderly Bevacizumab-Treated Patients with Metastatic Colorectal Cancer

Background

Studies suggest a relationship between hypertension and outcome in bevacizumab-treated patients with metastatic colorectal cancer (mCRC). We performed a retrospective analysis of two phase II studies (BECA and BECOX) to determine if hypertension and proteinuria predict outcome in elderly patients with mCRC treated with bevacizumab.

Patients and Methods

Patients ≥70 years of age received either capecitabine 1250 mg/m² bid days 1–14 + bevacizumab 7.5 mg/kg day 1 every 21 days (BECA study) or capecitabine 1000 mg/m² bid days 1–14 with bevacizumab 7.5 mg/kg and oxaliplatin 130 mg/m² day 1 (BECOX study). The primary objective was to correlate hypertension and proteinuria with overall response rate (ORR), time to progression (TTP) and overall survival (OS). Secondary objectives included identification of risk factors associated with the development of hypertension and proteinuria and determining whether development of hypertension or proteinuria in the first 2 cycles was related to ORR, disease-control rate (DCR), TTP or OS.

Results

In total, 127 patients (median age 75.5 years) were included in the study. Hypertension correlated with DCR and OS; proteinuria correlated with ORR and DCR. Proteinuria or hypertension in the first 2 cycles did not correlate with efficacy. Risk factors for hypertension were female gender (odds ratio [OR] 0.241; P = 0.011) and more bevacizumab cycles (OR 1.112; P = 0.002); risk factors for proteinuria were diabetes (OR 3.869; P = 0.006) and more bevacizumab cycles (OR 1.181; P<0.0001). Multivariate analysis identified as having prognostic value: baseline lactate dehydrogenase, haemoglobin, number of metastatic lesions and DCR.

Conclusion

This analysis of two phase II studies suggests that hypertension is significantly correlated with OS but not with ORR and TTP, whereas proteinuria is correlated with ORR but not with OS and TTP. Both hypertension and proteinuria are associated with the duration of bevacizumab treatment and do not represent an independent prognostic factor.     

Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L

The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, K _i = 2.35·10⁻⁸ M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (K _i = 1.3·10⁻⁹ M, and K _i = 1.2·10⁻⁸ M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor’s P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases.     

Maternal Mortality in Colombia in 2011: A Two Level Ecological Study

Objective

Maternal mortality reduction is a Millennium Development Goal. In Colombia, there is a large disparity in the maternal mortality ratio (MMR) between and into departments (states) and also between municipalities. We examined socioeconomics variables at the municipal and departmental levels which could be associated to the municipal maternal mortality in Colombia.

Methods

A multilevel ecology study was carried out using different national data sources in Colombia. The outcome variable was the MMR at municipal level in 2011 with multidimensional poverty at municipal and department level as the principal independent variables and other measures of the social and economic characteristics at municipal and departmental level were also considered explicative variables (overall fertility municipal rate, percentage of local rural population, health insurance coverage, per capita territorial participation allocated to the health sector, transparency index and Gini coefficient). The association between MMR and socioeconomic contextual conditions at municipal and departmental level was assessed using a multilevel Poisson regression model.

Results

The MMR in the Colombian municipalities was associated significantly with the multidimensional poverty (relative ratio of MMR: 3.52; CI 95%: 1.09-11.38). This association was stronger in municipalities from departments with the highest poverty (relative ratio of MMR: 7.14; CI 95%: 2.01-25.35). Additionally, the MMR at municipal level was marginally associated with municipally health insurance coverage (relative ratio of MMR: 0.99; CI 95%: 0.98-1.00), and significantly with transparency index at departmental level (relative ratio of MMR: 0.98; CI 95%: 0.97-0.99).

Conclusion

Poverty and transparency in a contextual level were associated with the increase of the municipal MMR in Colombia. The results of this study are useful evidence for informing the public policies discussion and formulation processes with a differential approach.     

Production of trichodiene by Trichoderma harzianum alters the perception of this biocontrol strain by plants and antagonized fungi

Trichothecenes are phytotoxic sesquiterpenic mycotoxins that can act as virulence factors in plant diseases. Harzianum A (HA) is a non‐phytotoxic trichothecene produced by Trichoderma arundinaceum. The first step in HA biosynthesis is the conversion of farnesyl diphosphate to trichodiene (TD), a volatile organic compound (VOC), catalysed by a sesquiterpene synthase encoded by the tri5 gene. Expression of tri5 in the biocontrol strain Trichoderma harzianum CECT 2413 resulted in production of TD in parallel with a reduction of ergosterol biosynthesis and an unexpected increase in the level of squalene. Transformants expressing tri5 displayed low chitinase activity and induced expression of Botrytis cinerea BOT genes, although their total antagonistic potential against phytopathogenic fungi was not reduced. VOCs released by the tri5‐transformant induced expression of tomato defence genes related to salicylic acid (SA), and TD itself strongly induced the expression of SA‐responsive genes and reduced the development of lateral roots. Together, these results suggest that TD acts as a signalling VOC in the interactions of Trichoderma with plants and other microorganisms by modulating the perception of this fungus to a given environment. Moreover, the TD ability to induce systemic defences indicates that complex trichothecene structures may not be necessary for inducing such responses.