On the geometrically exact low-order modelling of a flexible beam: formulation and numerical tests.

This paper proposes a low-order geometrically exact flexible beam formulation based on the utilization of generic beam shape functions to approximate distributed kinematic properties of the deformed structure. The proposed nonlinear beam shapes approach is in contrast to the majority of geometrically nonlinear treatments in the literature in which element-based-and hence high-order-discretizations are adopted. The kinematic quantities approximated specifically pertain to shear and extensional gradients as well as local orientation parameters based on an arbitrary set of globally referenced attitude parameters. In developing the dynamic equations of motion, an Euler angle parametrization is selected as it is found to yield fast computational performance. The resulting dynamic formulation is closed using an example shape function set satisfying the single generic kinematic constraint. The formulation is demonstrated via its application to the modelling of a series of static and dynamic test cases of both simple and non-prismatic structures; the simulated results are verified using MSC Nastran and an element-based intrinsic beam formulation. Through these examples, it is shown that the nonlinear beam shapes approach is able to accurately capture the beam behaviour with a very minimal number of system states.

E-orthodontics 2002.

The orthodontic specialty in Australia and New Zealand is at the forefront of the profession in evidence basing, efficacy and education, but cannot rest on its laurels. The future will require further changes in each area. Changes in evidence basing and education (particularly mandatory CPD) are likely to be government driven in the coming decade. Changes in efficacy may well be the area of most dramatic change in the medium term future and, as always, will be driven by the profession and technology.

Histone H3 specific acetyltransferases are essential for cell cycle progression.

Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G(2)/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities.

Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1.

Posttranslational modifications of histone amino termini play an important role in modulating chromatin structure and function. Lysine methylation of histones has been well documented, and recently this modification has been linked to cellular processes involving gene transcription and heterochromatin assembly. However, the existence of arginine methylation on histones has remained unclear. Recent discoveries of protein arginine methyltransferases, CARM1 and PRMT1, as transcriptional coactivators for nuclear receptors suggest that histones may be physiological targets of these enzymes as part of a poorly defined transcriptional activation pathway. Here we show by using mass spectrometry that histone H4, isolated from asynchronously growing human 293T cells, is methylated at arginine 3 (Arg-3) in vivo. In support, a novel antibody directed against histone H4 methylated at Arg-3 independently demonstrates the in vivo occurrence of this modification and reveals that H4 Arg-3 methylation is highly conserved throughout eukaryotes. Finally, we show that PRMT1 is the major, if not exclusive, H4 Arg-3 methyltransfase in human 293T cells. These findings suggest a role for arginine methylation of histones in the transcription process.

Cognitive precedence for local information in hierarchical stimulus processing by pigeons.

Four experiments investigated the processing of hierarchical stimuli by pigeons. Using a 4 alternative divided-attention task, 4 pigeons were food-reinforced for accurately identifying letters arranged as either hierarchical global- or local-relevant stimuli or as size-matched filled stimuli. Experiment 1 found that task acquisition was faster with local-relevant than global-relevant stimuli. This difference was not due to letter size. Experiment 2 demonstrated successful transfer to a novel irrelevant letter configuration. Experiments 3 and 4 tested pigeons' responses to conflict probe stimuli composed of equally discriminable relevant letters at each level. These tests revealed that all of the pigeons showed a cognitive precedence for local information early in processing, with the pigeons using different cues to initiate the processing of global information. This local advantage contrasts with previously reported results for humans and pigeons but is similar to that reported for nonhuman primates. Alternatives attempting to reconcile these contrasting comparative results are considered.

Identification and characterization of the carboxyl-terminal region of rat dentin sialoprotein.

Two acidic proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), are present in the extracellular matrix of dentin but not in bone. These two proteins are expressed in odontoblasts and preameloblasts as a single cDNA transcript coding a large precursor protein termed dentin sialophosphoprotein (DSPP). DSPP is specifically cleaved into two unique proteins, DSP and DPP. However, the cleavage site(s) of DSPP and the mechanisms for regulating the cleavages are unknown. To identify the specific site(s) of DSPP that are cleaved when the initial translation product is converted to DSP and DPP, we performed a detailed analysis (Edman degradation and mass spectrometry) on selected tryptic peptides of a size originating from the COOH-terminal region of rat DSP. After cleavage with trypsin, the DSP fragments were separated by a two-dimensional method (size-exclusion chromatography followed by reversed phase high performance liquid chromatography). We characterized 13 peptides from various regions of DSP. The analyses showed that peptide Ile(409)-Tyr(421) was the major COOH-terminal fragment, ending at Tyr(421) only 9 residues from the NH(2) terminus of DPP. Peptide Gln(385)-His(406) represented a second, minor COOH-terminal peptide that terminated at His(406). Both of these residues are well beyond the COOH terminus predicted previously by two independent studies estimating that rat DSP contained 360-370 amino acids. Careful studies on two peptides showed that, among 9 potential casein kinase II phosphorylation sites, 2 serines were phosphorylated. We found that rat DSP was heterogeneous with respect to phosphorylation, because this same peptide sequence eluted in two discrete peaks, one with 2 phosphoserines and the other having 1. The finding that 3 lysines just preceding the COOH termini were modified by a 43-Da substituent (possibly a carbamoyl substituent) suggests that the lysines in this region were particularly susceptible to attachment of this substituent.

Identification of a phosphorylation site in the hinge region of the human progesterone receptor and additional amino-terminal phosphorylation sites.

We have previously reported the identification of seven in vivo phosphorylation sites in the amino-terminal region of the human progesterone receptor (PR). From our previous in vivo studies, it was evident that several phosphopeptides remained unidentified. In particular, we wished to determine whether human PR contains a phosphorylation site in the hinge region, as do other steroid receptors including chicken PR, human androgen receptor, and mouse estrogen receptor. Previously, problematic trypsin cleavage sites hampered our ability to detect phosphorylation sites in large incomplete tryptic peptides. Using a combination of mass spectrometry and in vitro phosphorylation, we have identified six previously unidentified phosphorylation sites in human PR. Using nanoelectrospray ionization mass spectrometry, we have identified two new in vivo phosphorylation sites, Ser(20) and Ser(676), in baculovirus-expressed human PR. Ser(676) is analogous to the hinge site identified in other steroid receptors. Additionally, precursor ion scans identified another phosphopeptide that contains Ser(130)-Pro(131), a likely candidate for phosphorylation. In vitro phosphorylation of PR with Cdk2 has revealed five additional in vitro Cdk2 phosphorylation sites: Ser(25), Ser(213), Thr(430), Ser(554), and Ser(676). At least two of these, Ser(213) and Ser(676), are authentic in vivo sites. We confirmed the presence of the Cdk2-phosphorylated peptide containing Ser(213) in PR from in vivo labeled T47D cells, indicating that this is an in vivo site. Our combined studies indicate that most, if not all, of the Ser-Pro motifs in human PR are sites for phosphorylation. Taken together, these data indicate that the phosphorylation of PR is highly complex, with at least 14 phosphorylation sites.

Dynamic object perception by pigeons: discrimination of action in video presentations.

Two experiments examined the discrimination by pigeons of relative motion using computer-generated video stimuli. Using a go/no-go procedure, pigeons were tested with video stimuli in which the camera's perspective went either "around" or "through" an approaching object in a semi-realistic context. Experiment 1 found that pigeons could learn this discrimination and transfer it to videos composed from novel objects. Experiment 2 found that the order of the video's frames was critical to the discrimination of the videos. We hypothesize that the pigeons perceived a three-dimensional representation of the objects and the camera's relative motion and used this as the primary basis for discrimination. It is proposed that the pigeons might be able to form generalized natural categories for the different kinds of motions portrayed in the videos.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans.

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

The A-type potassium channel Kv4.2 is a substrate for the mitogen-activated protein kinase ERK.

The mitogen-activated protein kinase ERK has recently become a focus of studies of synaptic plasticity and learning and memory. Due to the prominent role of potassium channels in regulating the electrical properties of membranes, modulation of these channels by ERK could play an important role in mediating learning-related synaptic plasticity in the CNS. Kv4.2 is a Shal-type potassium channel that passes an A-type current and is localized to dendrites and cell bodies in the hippocampus. The sequence of Kv4.2 contains several consensus sites for ERK phosphorylation. In the present studies, we tested the hypothesis that Kv4.2 is an ERK substrate. We determined that the Kv4.2 C-terminal cytoplasmic domain is an effective ERK2 substrate, and that it is phosphorylated at three sites: Thr(602), Thr(607), and Ser(616). We used this information to develop antibodies that recognize Kv4.2 phosphorylated by ERK2. One of our phospho-site-selective antibodies was generated using a triply phosphorylated peptide as the antigen. We determined that this antibody recognizes ERK-phosphorylated Kv4.2 in COS-7 cells transfected with Kv4.2 and native ERK-phosphorylated Kv4.2 in the rat hippocampus. These observations indicate that Kv4.2 is a substrate for ERK in vitro and in vivo, and suggest that ERK may regulate potassium-channel function by direct phosphorylation of the pore-forming alpha subunit.

Stimulus repetition effects on texture-based visual search by pigeons.

Four experiments investigated the effects of within-session stimulus repetition on texture discrimination. Six pigeons (Columba livia) searched for a contrasting target region (color or shape) randomly embedded within a larger distractor region for food reinforcement. Experiment 1 found that repeating features of the distractors, but not those of the target, across trials increased the accuracy of target localization relative to baseline. Experiment 2 found that subsequently switching the identity of a repeated distractor feature to the target decreased accuracy. Experiment 3 found that the effects of repeating a distractor feature influenced search performance for at least 60 trials after this learning. Experiment 4 found that differential stimulus-outcome relations can produce control by repeated target features. The results are discussed in terms of the factors and strategies involved in the control of avian visual search behavior.

Kv4.2 phosphorylation by cyclic AMP-dependent protein kinase.

Recent evidence suggests that K(+) channels composed of Kv4.2 alpha-subunits underlie a transient current in hippocampal CA1 neurons and ventricular myocytes, and activation of the cAMP second messenger cascade has been shown to modulate this transient current. We determined if Kv4.2 alpha-subunits were directly phosphorylated by cAMP-dependent protein kinase (PKA). The intracellular domains of the amino and carboxyl termini of Kv4.2 were expressed as glutathione S-transferase fusion protein constructs; we observed that both of these fusion proteins were substrates for PKA in vitro. By using phosphopeptide mapping and amino acid sequencing, we identified PKA phosphorylation sites on the amino- and carboxyl-terminal fusion proteins corresponding to Thr(38) and Ser(552), respectively, within the Kv4.2 sequence. Kinetic characterization of the PKA sites demonstrated phosphorylation kinetics comparable to Kemptide. To evaluate PKA site phosphorylation in situ, phospho-selective antisera for each of the sites were generated. By using COS-7 cells expressing an EGFP-Kv4.2 fusion protein, we observed that stimulation of the endogenous PKA cascade resulted in an increase in phosphorylation of Thr(38) and Ser(552) within Kv4.2 in the intact cell. We also observed modulation of PKA phosphorylation at these sites within Kv4.2 in hippocampal area CA1. These results provide insight into likely sites of regulation of Kv4.2 by PKA.

The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation.

In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.

Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena.

Studies into posttranslational modifications of histones, notably acetylation, have yielded important insights into the dynamic nature of chromatin structure and its fundamental role in gene expression. The roles of other covalent histone modifications remain poorly understood. To gain further insight into histone methylation, we investigated its occurrence and pattern of site utilization in Tetrahymena, yeast, and human HeLa cells. In Tetrahymena, transcriptionally active macronuclei, but not transcriptionally inert micronuclei, contain a robust histone methyltransferase activity that is highly selective for H3. Microsequence analyses of H3 from Tetrahymena, yeast, and HeLa cells indicate that lysine 4 is a highly conserved site of methylation, which to date, is the major site detected in Tetrahymena and yeast. These data document a nonrandom pattern of H3 methylation that does not overlap with known acetylation sites in this histone. In as much as H3 methylation at lysine 4 appears to be specific to macronuclei in Tetrahymena, we suggest that this modification pattern plays a facilitatory role in the transcription process in a manner that remains to be determined. Consistent with this possibility, H3 methylation in yeast occurs preferentially in a subpopulation of H3 that is preferentially acetylated.

Dynamic object perception by pigeons.

Three experiments examined pigeon discrimination of computer-generated three-dimensional (3-D) projections of cube and pyramid objects. Four pigeons were tested using a go/no-go procedure involving static and dynamically rotating presentations of these stimuli. Transfer tests with different types of rotational and featural transformations suggested the pigeons may have used a 3-D representation of the objects as their primary means of performing the discrimination. The comparative implications for object and motion perception in animals are considered.

Identification and mutation of phosphorylation sites in a linker histone. Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena.

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.

Expanded lysine acetylation specificity of Gcn5 in native complexes.

The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila.

Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.