Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPgammaS

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K(12) of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido)triphosphate or guanosine-5'-(beta,gamma-methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) leads to (31)P NMR spectra that indicate the prevalence of only one conformational state with K(12) > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTPgammaS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affinity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy DeltaH(|) (1a1b) of 61 kJ.mol(-1) and an activation entropy DeltaS(|) (1a1b) of 65 J.K(-1).mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K(A) for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTPgammaS complex.     

Impact of ramp-up on the optimal capacity-related reconfiguration policy

This paper presents an optimal solution, based on Markov decision theory, for the problem of optimal capacity-related reconfiguration of manufacturing systems, under stochastic market demand. Both capacity expansion and reduction are considered. The solution quantitatively takes into account the effect of the ramp-up phenomenon, following each reconfiguration, on the optimal policy. A closed-form solution is presented for when product demand is independently and generally distributed over time. A real case concerning a flexible manufacturing line in the automotive sector is shown, to prove that ignoring the ramp-up effect in the decision process can lead to significant increases in overall costs.     

Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography

As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC.     

DDD: Distributed Dataset DNS

Cluster Computing - The advent of inexpensive data storage has resulted in larger and larger datasets as the cost of pruning data becomes more expensive then storing it for future insights. This...     

Follow-up assessment of patients with Pure Neural Leprosy in a reference center in Rio de Janeiro—Brazil

IntroductionPure Neural Leprosy (PNL) is a rare clinical form of leprosy in which patients do not present with the classical skin lesions but have a high burden of the disability associated with the disease. Clinical characteristics and follow up of patients in PNL are still poorly described in the literature.ObjectiveThis paper aims to describe the clinical, electrophysiological and histopathological characteristics of PNL patients, as well as their evolution after multidrug therapy (MDT).MethodsFifty-two PNL patients were selected. Clinical, nerve conduction studies (NCS), histopathological and anti-PGL-1serology were evaluated. Patients were also assessed monthly during the MDT. At the end of the MDT, all of the patients had a new neurological examination and 44 were submitted to another NCS.ResultsParesthesia was the complaint most frequently reported by patients, and in the neurological examination the most common pattern observed was impairment in sensory and motor examination and a mononeuropathy multiplex. Painful nerve enlargement, a classical symptom of leprosy neuropathy, was observed in a minority of patients and in the motor NCS axonal injuries, alone or in combination with demyelinating features, were the most commonly observed. 88% of the patients did not present any leprosy reaction during MDT. There was no statistically significant difference between the neurological examinations, nor the NCS pattern, performed before and after the MDT.DiscussionThe classical hallmarks of leprosy neuropathy are not always present in PNL making the diagnosis even more challenging. Nerve biopsy is an important tool for PNL diagnosis as it may guide therapeutic decisions. This paper highlights unique characteristics of PNL in the spectrum of leprosy in an attempt to facilitate the diagnosis and management of these patients.     

Live-imaging provides an atlas of cellular growth dynamics in the stamen

Development of multicellular organisms is a complex process involving precise coordination of growth among individual cells. Understanding organogenesis requires measurements of cellular behaviors over space and time. In plants, such a quantitative approach has been successfully used to dissect organ development in both leaves and external floral organs, such as sepals. However, the observation of floral reproductive organs is hampered as they develop inside tightly closed floral buds, and are therefore difficult to access for imaging. We developed a confocal time-lapse imaging method, applied here to Arabidopsis (Arabidopsis thaliana), which allows full quantitative characterization of the development of stamens, the male reproductive organs. Our lineage tracing reveals the early specification of the filament and the anther. Formation of the anther lobes is associated with a temporal increase of growth at the lobe surface that correlates with intensive growth of the developing locule. Filament development is very dynamic and passes through three distinct phases: (1) initial intense, anisotropic growth, and high cell proliferation; (2) restriction of growth and proliferation to the filament proximal region; and (3) resumption of intense and anisotropic growth, displaced to the distal portion of the filament, without cell proliferation. This quantitative atlas of cellular growth dynamics provides a solid framework for future studies into stamen development.     

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.