Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. II. Tripeptides with short side chains populating asx and β‐type like turn conformations

In the preceding paper, we found that ensembles of tripeptides with long or bulky chains can include up to 20% of various turns. Here, we determine the structural and thermodynamic characteristics of GxG peptides with short polar and/or ionizable central residues (D, N, C), whose conformational distributions exhibit higher than average percentage (>20%) of turn conformations. To probe the side‐chain conformations of these peptides, we determined the ³J(H^α,H^β) coupling constants and derived the population of three rotamers with χ₁‐angles of ?60°, 180° and 60°, which were correlated with residue propensities by DFT‐calculations. For protonated GDG, the rotamer distribution provides additional evidence for asx‐turns. A comparison of vibrational spectra and NMR coupling constants of protonated GDG, ionized GDG, and the protonated aspartic acid dipeptide revealed that side chain protonation increases the pPII content at the expense of turn populations. The charged terminal groups, however, have negligible influence on the conformational properties of the central residue. Like protonated GDG, cationic GCG samples asx‐turns to a significant extent. The temperature dependence of the UVCD spectra and ³J(H^NH^α) constants suggest that the turn populations of GDG and GNG are practically temperature‐independent, indicating enthalpic and entropic stabilization. The temperature‐independent J‐coupling and UVCD spectra of GNG require a three‐state model. Our results indicate that short side chains with hydrogen bonding capability in GxG segments of proteins may serve as hinge regions for establishing compact structures of unfolded proteins and peptides. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

The Supplementary Motor Area Exerts a Tonic Excitatory Influence on Corticospinal Projections to Phrenic Motoneurons in Awake Humans

Introduction

In humans, cortical mechanisms can interfere with autonomic breathing. Respiratory-related activation of the supplementary motor area (SMA) has been documented during voluntary breathing and in response to inspiratory constraints. The SMA could therefore participate in the increased resting state of the respiratory motor system during wake (i.e. "wakefulness drive to breathe").

Methods

The SMA was conditioned by continuous theta burst magnetic stimulation (cTBS, inhibitory) and 5 Hz conventional rTMS (5 Hz, excitatory). The ensuing effects were described in terms of the diaphragm motor evoked response (DiMEPs) to single-pulse transcranial magnetic stimulation over the motor cortex. DiMEPs were recorded at baseline, and at 3 time-points ("post1", "post2", "post3") up to 15 minutes following conditioning of the SMA.

Results

cTBS reduced the amplitude of DiMEPs from 327.5±159.8 µV at baseline to 243.3±118.7 µV, 217.8±102.9 µV and 240.6±123.9 µV at post 1, post 2 and post 3, respectively (F = 6.341, p = 0.002). 5 Hz conditioning increased the amplitude of DiMEPs from 184.7±96.5 µV at baseline to 270.7±135.4 µV at post 3 (F = 4.844, p = 0.009).

Conclusions

The corticospinal pathway to the diaphragm can be modulated in both directions by conditioning the SMA. This suggests that the baseline respiratory activity of the SMA represents an equipoise from which it is possible to move in either direction. The resting corticofugal outflow from the SMA to phrenic motoneurones that this study evidences could putatively contribute to the wakefulness drive to breathe.     

Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues

To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.     

Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.     

Dynamics and differential proliferation of transposable elements during the evolution of the B and A genomes of wheat

Transposable elements (TEs) constitute >80% of the wheat genome but their dynamics and contribution to size variation and evolution of wheat genomes (Triticum and Aegilops species) remain unexplored. In this study, 10 genomic regions have been sequenced from wheat chromosome 3B and used to constitute, along with all publicly available genomic sequences of wheat, 1.98 Mb of sequence (from 13 BAC clones) of the wheat B genome and 3.63 Mb of sequence (from 19 BAC clones) of the wheat A genome. Analysis of TE sequence proportions (as percentages), ratios of complete to truncated copies, and estimation of insertion dates of class I retrotransposons showed that specific types of TEs have undergone waves of differential proliferation in the B and A genomes of wheat. While both genomes show similar rates and relatively ancient proliferation periods for the Athila retrotransposons, the Copia retrotransposons proliferated more recently in the A genome whereas Gypsy retrotransposon proliferation is more recent in the B genome. It was possible to estimate for the first time the proliferation periods of the abundant CACTA class II DNA transposons, relative to that of the three main retrotransposon superfamilies. Proliferation of these TEs started prior to and overlapped with that of the Athila retrotransposons in both genomes. However, they also proliferated during the same periods as Gypsy and Copia retrotransposons in the A genome, but not in the B genome. As estimated from their insertion dates and confirmed by PCR-based tracing analysis, the majority of differential proliferation of TEs in B and A genomes of wheat (87 and 83%, respectively), leading to rapid sequence divergence, occurred prior to the allotetraploidization event that brought them together in Triticum turgidum and Triticum aestivum, <0.5 million years ago. More importantly, the allotetraploidization event appears to have neither enhanced nor repressed retrotranspositions. We discuss the apparent proliferation of TEs as resulting from their insertion, removal, and/or combinations of both evolutionary forces.     

Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1α,25(OH)₂D₃can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized ³H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1α,25(OH)₂D₃ analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2α- or 2β-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1α,25(OH)₂D₃. The ³H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1α,25(OH)₂D₃, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ or 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ for 7 days. 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ and 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ were also shown to be about 10-fold more active than 1α,25(OH)₂D₃ in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1α,25(OH)₂D₃ molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.     

New palaeontological assemblage, sedimentological and chronological data from the Pleistocene Ma U'Oi cave (northern Vietnam)

This paper describes recent material gathered during the second fieldwork at Ma U'Oi in November 2002 by a Vietnamese–French–Japanese team. The Ma U'Oi cave, located in the province of Hoà Binh (60 km SW from Hanoi), northern Vietnam, belongs to a karstic network developed in Triassic dark-grey limestones.

The cave is filled with coarse-grained breccias containing numerous fossil remains, partially preserved at several loci inside the cave (wall, vault and ground). We describe new teeth which confirm the occurrence of mammal taxa already mentioned at Ma U'Oi (Bacon et al., 2004)[Bacon, A-M., Demeter, F., Schuster, M., Long, V.T., Thuy, N.K., Antoine, P-O., Sen, S., Nga, H.H., Huong, N.T.M., 2004. The Pleistocene Ma U'Oi cave, northern Vietnam: palaeontology, sedimentology and palaeoenvironments. Geobios 37, 305–314], while others, mainly microvertebrates, emphasize the occurrence of new species for the Pleistocene of Vietnam. We report here, for the first time, the occurrence of these microvertebrates of different groups (primates, rodents, insectivores, small reptiles and amphibians) in the faunal assemblage. Among mammal taxa, the presence of one more hominid affiliated to archaic Homo is also attested by our findings. U/Th dating carried out on 2 samples extracted from breccia speleothems confirms the biochronological estimate, with fossiliferous fillings ranging from late Middle Pleistocene to Late Pleistocene.