Axial loading induces rotation of the proximal carpal row bones around unique screw-displacement axes

The changes in carpal bone alignment secondary to the aplication of an axial compressive load through the major wrist motor tendons while the wrist is kept in neutral position (isometric loading) have been investigated on 13 fresh cadaver specimens using a biplanar radiographic method of kinematic analysis. The scaphoid, lunate and triquetrum rotate an average of 5.1, 4.2 and 3.8°, respectively, around different ‘screw displacement axes’, all implying flexion, radial deviation and supination. Based on these findings, a new interpretation of the mechanism by which the wrist remains stable under physiologic loads is provided.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV‐1 co‐infections. We have delineated the mechanism of cell death induced by the HIV‐1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir–Leishmania interactions, we selected Nelfinavir‐resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir‐resistant and ‐sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir‐resistant and ‐sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time‐dependent manner. Furthermore, high‐resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir‐resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug‐induced intracellular vesicles.     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Frequency Control in Synchronized Networks of Inhibitory Neurons

We analyze the control of frequency for a synchronized inhibitory neuronal network. The analysis is done for a reduced membrane model with a biophysically based synaptic influence. We argue that such a reduced model can quantitatively capture the frequency behavior of a larger class of neuronal models. We show that in different parameter regimes, the network frequency depends in different ways on the intrinsic and synaptic time constants. Only in one portion of the parameter space, called phasic, is the network period proportional to the synaptic decay time. These results are discussed in connection with previous work of the authors, which showed that for mildly heterogeneous networks, the synchrony breaks down, but coherence is preserved much more for systems in the phasic regime than in the other regimes. These results imply that for mildly heterogeneous networks, the existence of a coherent rhythm implies a linear dependence of the network period on synaptic decay time and a much weaker dependence on the drive to the cells. We give experimental evidence for this conclusion.     

Four differently chromatin‐associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific β-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their ‘extractability’ from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.     

Impact of Feedback Phosphorylation and Raf Heterodimerization on Normal and Mutant B-Raf Signaling

The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.The Ras, Raf, MEK, and extracellular signal-regulated kinase (ERK) proteins are core components of one of the major signaling cascades regulating normal cell proliferation—the Ras pathway. Not surprising, deregulation of Ras pathway signaling is a major contributor to human cancer and has recently been linked with several developmental disorders, such as Noonan''s, LEOPARD, and cardiofaciocutaneous (CFC) syndromes (28). Given its importance to both normal and disease states, much effort has been directed toward elucidating the mechanisms that modulate Ras pathway signaling. Of all the pathway components, regulation of the Raf proteins has proved to be the most complex, involving inter- and intramolecular interactions, a change in subcellular localization, and phosphorylation and dephosphorylation events (6, 32).In mammalian cells, there are three Raf family members: A-Raf, B-Raf, and C-Raf (12). In their inactive state, all Raf proteins are found in the cytosol, with the N-terminal regulatory domain acting as an autoinhibitor of the C-terminal kinase domain (4, 5, 13). 14-3-3 dimers bind to phosphorylation sites present in both the N- and C-terminal regions and stabilize the autoinhibited state (22). To activate the Raf proteins, autoinhibition mediated by the N terminus must be relieved and the kinase domain must adopt the active catalytic conformation (6, 31, 32). Under normal signaling conditions, Ras activation helps mediate these events by recruiting the Raf proteins to the plasma membrane, which induces the release of 14-3-3 from the N-terminal binding site and facilitates phosphorylation of the Raf kinase domain (19). For the C-Raf and A-Raf proteins, phosphorylation occurs in two regions of the kinase domain, the negative-charge regulatory region (N-region) and the activation segment (4). In contrast, the N-region of B-Raf exhibits a constitutive negative charge due to increased basal phosphorylation of an activating serine site and the presence of two aspartic acid residues (18); thus, only phosphorylation of the activation segment is required. Phosphorylation of the activation segment serves both to destabilize the “inactive” catalytic conformation maintained by hydrophobic interactions between the glycine-rich loop and the activation segment and to stabilize the “active” catalytic conformation, whereas the negative charge of the N-region helps to disrupt the autoinhibitory activity of the N-terminal domain (5, 30, 31).Because the N-region of B-Raf exhibits a constitutive negative charge, B-Raf possesses higher basal kinase activity than other family members and is more susceptible to mutational activation (9, 11, 17). In particular, B-Raf is a major contributor to human cancer: somatic mutations in the B-Raf gene are detected in ∼50% of malignant melanomas and many colorectal, ovarian, and papillary thyroid carcinomas (7). Of the oncogenic mutations identified in B-Raf, the vast majority cluster to the two regions of the kinase domain responsible for maintaining the inactive catalytic conformation—the glycine-rich loop and the activation segment (31). Based on enzymatic activity, the oncogenic B-Raf proteins have been divided into three groups: those with high activity (130- to 700-fold more active than wild-type [WT] B-Raf), those with intermediate activity (64- to 1.3-fold more active), and surprisingly, those with impaired catalytic activity (0.8 to 0.3 of WT B-Raf activity) (31). Further analysis has revealed that all oncogenic B-Raf proteins heterodimerize constitutively with C-Raf and activate C-Raf in a Ras-independent manner that requires an intact C-Raf activation segment as well as the binding of 14-3-3 to the C-terminal pS621 binding site on C-Raf (11). Importantly, for the oncogenic B-Raf proteins with impaired kinase activity, the binding and activation of C-Raf are required for ERK activation in vivo (31). Interestingly, heterodimerization of B-Raf and C-Raf also occurs under normal signaling conditions; however, in this case, heterodimerization is Ras dependent and occurs at the plasma membrane following mitogen stimulation (11, 27).Once activated, either by upstream signaling or by mutational events, all Raf proteins are capable of initiating the phosphorylation cascade that results in the sequential activation of MEK and ERK. ERK then phosphorylates targets in both the cytoplasm and the nucleus that are required for cell proliferation. Strikingly, the Raf proteins themselves are also substrates of activated ERK. In regard to C-Raf, ERK-dependent feedback phosphorylation has been shown to instigate a regulatory cycle whereby phosphorylation of the feedback sites down-modulates C-Raf signaling, after which the hyperphosphorylated C-Raf protein is dephosphorylated and returned to a signaling-competent state through dephosphorylation events involving protein phosphatase 2A (PP2A) and the Pin1 prolyl-isomerase (8). For B-Raf, two ERK-dependent feedback sites, S750 and T753, have been identified, and phosphorylation of these sites has been reported to have a negative regulatory effect (3).In this study, we have further investigated the impact of feedback phosphorylation and heterodimerization on B-Raf signaling. Here we find that both normal and oncogenic B-Raf proteins are phosphorylated on four S/TP sites (S151, T401, S750, and T753) by activated ERK. Through mutational analysis, we find that phosphorylation of B-Raf at S151 inhibits binding to activated Ras, whereas phosphorylation of each of the feedback sites contributes to the disruption of B-Raf/C-Raf heterodimers. Moreover, we find that events influencing B-Raf/C-Raf heterodimerization, such as feedback phosphorylation and 14-3-3 binding, can alter the signaling activity of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity as well as that of B-Raf and C-Raf proteins containing mutations identified in CFC and Noonan''s syndromes, respectively.     

Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration

In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.     

Importance of factors determining the effective lifetime of a mass, long-lasting, insecticidal net distribution: a sensitivity analysis

Background

Long-lasting insecticidal nets (LLINs) reduce malaria transmission by protecting individuals from infectious bites, and by reducing mosquito survival. In recent years, millions of LLINs have been distributed across sub-Saharan Africa (SSA). Over time, LLINs decay physically and chemically and are destroyed, making repeated interventions necessary to prevent a resurgence of malaria. Because its effects on transmission are important (more so than the effects of individual protection), estimates of the lifetime of mass distribution rounds should be based on the effective length of epidemiological protection.

Methods

Simulation models, parameterised using available field data, were used to analyse how the distribution's effective lifetime depends on the transmission setting and on LLIN characteristics. Factors considered were the pre-intervention transmission level, initial coverage, net attrition, and both physical and chemical decay. An ensemble of 14 stochastic individual-based model variants for malaria in humans was used, combined with a deterministic model for malaria in mosquitoes.

Results

The effective lifetime was most sensitive to the pre-intervention transmission level, with a lifetime of almost 10 years at an entomological inoculation rate of two infectious bites per adult per annum (ibpapa), but of little more than 2 years at 256 ibpapa. The LLIN attrition rate and the insecticide decay rate were the next most important parameters. The lifetime was surprisingly insensitive to physical decay parameters, but this could change as physical integrity gains importance with the emergence and spread of pyrethroid resistance.

Conclusions

The strong dependency of the effective lifetime on the pre-intervention transmission level indicated that the required distribution frequency may vary more with the local entomological situation than with LLIN quality or the characteristics of the distribution system. This highlights the need for malaria monitoring both before and during intervention programmes, particularly since there are likely to be strong variations between years and over short distances. The majority of SSA's population falls into exposure categories where the lifetime is relatively long, but because exposure estimates are highly uncertain, it is necessary to consider subsequent interventions before the end of the expected effective lifetime based on an imprecise transmission measure.