Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1.

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.

Development of new hydroxamate matrix metalloproteinase inhibitors derived from functionalized 4-aminoprolines.

A series of hydroxamates was prepared from an aminoproline scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors, such as compound 47, display broad-spectrum activity with sub-nanomolar potency for some enzymes. Modifications of the P1' portion of the molecule played a key role in affecting both potency and selectivity within the MMP family. Longer-chain aliphatic substituents in this region of the molecule tended to increase potency for MMP-3 and decrease potency for MMP-1, as exemplified by compounds 48-50, while aromatic substituents, as in compound 52, generated broad-spectrum inhibition. The data is rationalized based upon X-ray crystal data which is also presented. While the in vitro peroral absorption seemed to be less predictable, it tended to decrease with longer and more hydrophilic substituents. Finally, a rat model of osteoarthritis was used to evaluate the efficacy of these compounds, and a direct link was established between their pharmacokinetics and their in vivo efficacy.

Development of new carboxylic acid-based MMP inhibitors derived from functionalized propargylglycines.

A series of carboxylic acids were prepared from a propargylglycine scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for four enzymes within the MMP family. The inhibitors were typically potent against collagenase-3 (MMP-13) and gelatinase A (MMP-2), while they spared collagenase-1 (MMP-1) and only moderately inhibited stromelysin (MMP-3). Compound 40 represents a typical inhibition profile of a compound with reasonable potency. Introduction of polar groups was required in order to generate inhibitors with acceptable water solubility, and this often resulted in a loss of potency as in compound 63. High serum protein binding proved to be a difficult hurdle with many compounds such as 48 showing >99% binding. Some compounds such as 64 displayed approximately 90% binding, but no reliable method was discovered for designing molecules with low protein binding. Finally, selected data regarding the pharmacokinetic behavior of these compounds is presented.

Real-time transmission of full-motion echocardiography over a high-speed data network: impact of data rate and network quality of service.

UNLABELLED: With high-resolution network transmission required for telemedicine, education, and guided-image acquisition, the impact of errors and transmission rates on image quality needs evaluation. METHODS: We transmitted clinical echocardiograms from 2 National Aeronautics and Space Administration (NASA) research centers with the use of Motion Picture Expert Group-2 (MPEG-2) encoding and asynchronous transmission mode (ATM) network protocol over the NASA Research and Education Network. Data rates and network quality (cell losses [CLR], errors [CER], and delay variability [CVD]) were altered and image quality was judged. RESULTS: At speeds of 3 to 5 megabits per second (Mbps), digital images were superior to those on videotape; at 2 Mbps, images were equivalent. Increasing CLR caused occasional, brief pauses. Extreme CER and CDV increases still yielded high-quality images. CONCLUSIONS: Real-time echocardiographic acquisition, guidance, and transmission is feasible with the use of MPEG-2 and ATM with broadcast quality seen above 3 Mbps, even with severe network quality degradation. These techniques can be applied to telemedicine and used for planned echocardiography aboard the International Space Station.

Diethylene glycol mono butyl ether concentrations in room air from application of cleaner formulations to hard surfaces.

Diethylene glycol monobutyl ether (DGBE) is a solvent used in some liquid hard surface cleaners. We evaluated the inhalation component of consumer exposure in the home to DGBE from the use of cleaning products containing up to 9% DGBE. Several experiments were conducted with restricted room air flow, exaggerated amounts of cleaning solutions, and no rinsing in order to develop an exposure scenario that would exceed exposures likely encountered by consumers. DGBE vapors in the air were monitored by collection on charcoal tubes, followed by desorption and quantitation by gas chromatography. Air was collected from the centre of the room and from the breathing zone of the person doing the washing task. Room air concentrations of DGBE showed peak values between one and three hours after task initiation; DGBE concentrations then gradually decreased with time. Peak concentrations did not exceed 1.6 ppmv. The total DGBE in the air at the time of maximum air concentrations accounted for only 1 to 3% of the DGBE on the washed surfaces. The person doing the washing task was exposed to average DGBE concentrations in the breathing zone below 0.8 ppmv in all experiments. The methods described for measuring DGBE concentrations in air are generally applicable to other solvents and easily adaptable to various experimental situations.

Electrical stimulation with bone and wound healing.

Electrical stimulation has been used to heal fractures and ulcers and reduce pain through modulation of local body processes. It has been recognized that mechanical forces and bioelectricity have an intimate relationship in influencing the production of bone. Science has developed techniques to affect change in the electrical charge of fractures to positively affect the healing process. Electrical stimulation, through invasive and noninvasive applications, has produced excellent results in the treatment of nonunions and ulcer care. A thorough review of the electrical properties of bone and soft tissue and the influence of electrical stimulation on healing is presented here.

Centromere identity, function, and epigenetic propagation across cell divisions.

The key to understanding centromere identity is likely to lie in the chromatin containing the histone H3 variant CENP-A. CENP-A is the prime candidate to carry the epigenetic information that specifies the chromosomal location of the centromere in nearly all eukaryotic species, raising questions fundamental to understanding chromosome inheritance: How is the epigenetic centromere mark propagated? What physical properties of CENP-A-containing complexes are important for epigenetically marking centromeres? What are the molecules that recognize centromeric chromatin and serve as the foundation for the mitotic kinetochore? We discuss recent advances from our research groups that have yielded substantial insight into these questions and present our current understanding of the centromere. Future work promises an understanding of the molecular processes that confer fidelity to genome transmission at cell division.

Combined mitochondrial and nuclear sequences support the monophyly of forcipulatacean sea stars.

Previous molecular phylogenetic analyses of forcipulatacean sea stars (Echinodermata: Asteroidea) have reconstructed a non-monophyletic order Forcipulatida, provided that two or more forcipulate families are included. This result could mean that one or more assumptions of the reconstruction method was violated, or else the traditional classification could be erroneous. The present molecular phylogenetic analysis included 12 non-forcipulatacean and 39 forcipulatacean sea stars, with multiple representatives of all but one of the forcipulate families and/or subfamilies. Bayesian analysis of approximately 4.2kb of sequence data representing seven partitions (nuclear 18S rRNA and 28S rRNA, mitochondrial 12S rRNA, 16S rRNA, 5 tRNAs and cytochrome oxidase I with first and second codon positions analyzed separately from third codon positions) recovered a consensus tree with three well-supported clades (78%-100% bootstrap support) that corresponded at least approximately to traditional taxonomic ranks: the superorder Forcipulatacea (Forcipulatida + Brisingida) + Pteraster, the Brisingida/Brisingidae and Asteriidae + Rathbunaster + Pycnopodia. When a molecular clock was enforced, the partitioned Bayesian analysis recovered the traditional Forcipulatacea. Five of six genera represented by two or more species were monophyletic with 100% bootstrap support. Most of the traditional subfamilial and familial groupings within the Forcipulatida were either unresolved or non-monophyletic. The separate partitions differed considerably in estimates of model parameters, mainly between nuclear sequences (with high GC content, low rates of sequence substitution and high transition/transversion rate ratios) and mitochondrial sequences.

Segregation and linkage studies of allozyme loci in pair crosses of the oyster Crassostrea virginica.

The genetic control of 11 electrophoretically detected allozyme polymorphisms in the oyster Crassostrea virginica was investigated in 10 pair crosses. For nine allozyme loci, each offspring shared at least one band (electromorph) with each parent. For the remaining two loci (mannosephosphate isomerase and leucine aminopeptidase-2), some offspring failed to share a band with one or both parents. Several lines of evidence indicated that these anomalous results were due to transmission of null alleles. There was evidence of distorted segregation at 8 of the 11 loci. The departures from the Mendelian expectations within the pair crosses might be due either to viability selection in the offspring or to gametic selection in one or both parents, although the possibility that the distortion is due to a locus linked to the allozyme locus cannot be ruled out. However, there was no evidence that heterozygosity per se had an effect on viability of offspring within a cross. Linkage analysis revealed two linkage groups, one consisting of four allozyme loci and the other consisting of three loci.

Estimating the degree of sperm precedence in laboratory mating experiments: a maximum likelihood method.

Allozyme polymorphisms have been used frequently in laboratory mating experiments to study patterns of sperm utilization in multiply mated females. In some instances, due either to chance or to design, there is a diagnostic difference between male genotypes that allows unambiguous assignment of paternity. In other instances, there is some overlap in allelic composition of males, so that attribution of paternity is often uncertain. This paper presents a statistical method for analyzing data of the second sort obtained from twice-mated females, based on the principle of maximum likelihood. The method allows the estimation of a mating parameter, psi the frequency with which sperm from the first male fertilizes the female's eggs. Various hypotheses about the null value of psi may be tested by a likelihood ratio test statistic. Also presented is a method of testing for homogeneity in psi values across different broods produced by the same female.

Hyperphosphorylation and association with RBP of the intracellular domain of Notch1.

Although the intracellular domain of Notch1 is phosphorylated and it associates with members of the CSL family, the relationship of these events is poorly understood. Using in vivo [(32)P]orthophosphate labeling of cells expressing transfected Notch1, we observed that the furin cleaved Notch1 (TMIC) and the soluble intracellular forms (NICD), but not the full-length molecule were phosphorylated. Furthermore, transfected NICD molecules showed a significantly greater specific activity of phosphorylation, or hyperphosphorylation, compared to TMIC molecules. Hyperphosphorylation of NICD was also observed when NICD was generated by an endogenous intramembraneous cleavage of TMIC. However, TMIC molecules bearing a mutation that reduces intramembraneous cleavage (V1744K) did not show an enhanced incorporation of phosphate, suggesting that cleavage is required for hyperphosphorylation. Using deletion constructs to map the sites of phosphorylation, we observed that a domain of 93 amino acids downstream of the ankyrin repeats incorporated the majority of (32)P in vivo. This sequence was also required for activation of the HES-1 promoter. In addition, we observed that hyperphosphorylated forms of the intracellular domain were more likely to interact with the transcriptional coactivator RBP. However, dephosphorylation experiments showed that the interaction between RBP and the intracellular domain of Notch was not dependent upon Notch1IC phosphorylation. These studies reveal that phosphorylation of the intracellular domain of the Notch receptor is a dynamic process during the events of Notch signal transduction.

Mitochondrial DNA sequence variation in a sea star (Leptasterias spp.) species complex.

A 551-bp region of a PCR product containing the putative mitochondrial control region and flanking sequences was analyzed for sequence variation among 19 sea stars representing 10 previously described PCR-RFLP haplotypes within a cryptic species complex (Leptasterias spp.). Most (97%) of the sequence variation was interhaplotypic rather than intrahaplotypic, which greatly reduced the utility of sequence polymorphisms in this mtDNA region as markers of intrahaplotypic population structure and gene flow. The estimated number of transition and transversion substitutions per nucleotide site, corrected for multiple hits, was 0.0364 and 0.0158, respectively. Most of the sequence variation occurred in the first half of the putative control region. Phylogenetic analysis (both maximum parsimony and maximum likelihood) revealed three well-supported clades, but the position of two PCR-RFLP haplotypes was not completely resolved. Low intraspecific mtDNA sequence divergence over large geographic distances may be a general pattern for echinoderm species.

Genetics of scnDNA polymorphisms in juvenile oysters (Crassostrea virginica). Part I: Characterizing the inheritance of polymorphisms in controlled crosses.

The amplification of DNA by polymerase chain reaction followed by restriction enzyme digestion was used to examine five anonymous single-copy nuclear DNA polymorphisms in 11 pair crosses for juvenile oysters (Crassostrea virginica). There was an overall 7% frequency of aberrant (non-Mendelian) offspring genotypes among 174 total pair-cross progeny. Strict Mendelian inheritance of alleles was observed for two of the five loci. Possible explanations for the aberrant offspring genotypes include nonamplifiable alleles owing to priming site polymorphism or aneuploidy, or paralogous amplification. Analysis of more pair crosses is needed to test these explanations.

Decoupling of molecular and morphological evolution in deep lineages of a meiobenthic harpacticoid copepod.

Molecular and biochemical genetic analyses have revealed that many marine invertebrate taxa, including some well-studied and presumably cosmopolitan species, are actually complexes of sibling species. When morphological differences are slight and estimated divergence times are old, data suggest either unusually high rates of sequence evolution or long-term morphological stasis. Here, five gene regions (mitochondrial cytochrome oxidase subunit I and large-subunit ribosomal 16S rDNA and nuclear ITS1, 5.8S rDNA, and ITS2) were analyzed in four geographic samples of the meiobenthic harpacticoid copepod Cletocamptus deitersi. Molecular sequences revealed four extremely differentiated molecular lineages with unalignable nuclear intergenic spacers and mitochondrial uncorrected divergences reaching 25% (cytochrome oxidase) and 36% (16S rDNA). These levels of divergence are greater than those reported previously for congeneric species in diverse invertebrate taxa, including crustaceans. The nominally intraspecific divergence matches or exceeds the corresponding divergence from a known congener (Cletocamptus helobius). A molecular clock applied to the cytochrome oxidase subunit I data suggests that these lineages split in the Miocene, consistent with the fossil record of a North American Cletocamptus from the same period. Morphological differences among the major lineages are subtle but congruent with the patterns of genetic differentiation. Our conclusion, based on concordant patterns of variation in two mitochondrial and three nuclear gene regions, as well as morphological observations, is that C. deitersi in North America is composed of at least four separate species by the genealogical concordance, phylogenetic, and morphological-species criteria. Alternative explanations for the deep phylogenetic nodes and apparent morphological stasis, including high rates of sequence evolution, balancing selection, and genetic signatures of historical events, are considered unlikely.

Amplification dynamics of human-specific (HS) Alu family members.

We have investigated the distribution of several recently inserted Alu family members within representatives of diverse human groups. Human population studies using 65 unrelated human DNA samples, as well as a familial study to test inheritance, showed that individual Alu family members could be divided into three groups. The first group consisted of relatively older Alu family members which were monomorphic (homozygous) throughout the population tested (HS C3N1 and C4N6). The second group (HS C4N2, C4N5 and C4N8), apparently inserted into other repetitive regions of the genome, resulting in inconclusive results in the PCR test used. However, it is clear that these particular Alu insertions were present in a majority if not all of the loci tested. The third group was comprised of three dimorphic Alu family members (HS C2N4, C4N4 and TPA 25). Only a single Alu family member (TPA 25) displayed a high degree of dimorphism within the human population. This latter example also showed different allele frequencies in different human groups. The isolation and characterization of additional highly dimorphic Alu family members should provide a useful tool for human population genetics.

Automated 96-well SPE and LC-MS-MS for determination of protease inhibitors in plasma and cartilage tissues.

Bioanalytical methods based on automated solid-phase extraction (SPE) and high-performance liquid chromatography with electrospray tandem mass spectrometry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automated using a 96-well extraction plate and a 96-channel programmable liquid-handling workstation. The LC-MS-MS methods were developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma or ground cartilage supernatant samples were loaded onto an SPE plate to remove proteins and other interfering components in the matrixes to render relatively clean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection. The methods were validated and successfully applied to the analysis of protease inhibitors in plasma and cartilage tissues.