The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

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.     

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.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.     

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.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

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.