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PubMed1992

Early Caulobacter crescentus genes fliL and fliM are required for flagellar gene expression and normal cell division.

The biogenesis of the Caulobacter crescentus polar flagellum requires the expression of more than 48 genes, which are organized in a regulatory hierarchy. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. In addition to the motility phenotype, mutations in this locus also cause abnormal cell division. Complementing clones restore both motility and normal cell division. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. FliM is thought to be a switch protein and to interface with the flagellum motor. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Tn5 insertions in the fliM gene prevent the transcription of class II and class III flagellar genes, which are lower in the regulatory hierarchy. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Sequence comparison of the fliL transcription start site with those of other class I genes, flaS and flaO, revealed a highly conserved 29-bp sequence in a potential promoter region that differs from sigma 70, sigma 54, sigma 32, and sigma 28 promoter sequences, suggesting that at least three class I genes share a unique 5' regulatory region.

Amino Acid Sequence↗

PubMed1996

Spatial characterization of contracting cardiac myocytes by computer-assisted, video-based image processing.

The goals of the present study were to develop and validate a computer-assisted, video-based image processing (CAVIP) system to measure time-dependent changes in isolated myocyte geometry during contraction and to use the CAVIP system to examine spatial characteristics of the myocyte during contraction in normal myocytes and in myocytes after development of dilated cardiomyopathy (DCM). Myocytes were isolated from the left ventricles of five control pigs and five pigs that developed chronic tachycardia (240 beats/min; 3 wk)-induced DCM. Isolated myocytes were stimulated and recorded using a high-speed camera interfaced with a standard video recording system. There was a significant linear relation between the indexes of time-dependent changes in myocyte length as measured by a conventional video edge-detector system and by the CAVIP system (r > 0.96; P < 0.01). After this validation procedure, dynamic changes in myocyte width and profile area with DCM were examined. Myocyte resting profile area was 33% larger in DCM myocytes compared with controls. However, there was no difference in the rate of area change with contraction between the two groups. Percent changes in myocyte width and profile area at peak contraction were significantly lower in the DCM group (43 and 46% respectively, P < 0.05). Therefore, the present study demonstrated that the CAVIP system provides unique information on time-dependent changes in myocyte geometry during contraction, particularly with the development of cardiomyopathic disease.

Animals↗

PubMed1997

Experiences with computed radiography: can we afford the cost?

In summary, we believe that the CR system adds significantly to our department. The film quality is superb, and the learning curve for radiologists to adapt to the added noise from anatomic structures on chest radiographs is short. The inherent film latitude and flexibility add a new dimension to interpretation and film management. System costs are significantly greater than for traditional film-screen systems. Recurring operational costs are yet to be determined but certainly will be greater with the shorter expected life span of the imaging plates. Film cost savings cannot be a justifiable reason for using a CR system. Film cost savings must await the ultimate conversion to soft imaging through networks and workstations. Technical issues are significant and require structured and specific courses of study and workshops for users before implementation of the CR system. As CR owners, we were frustrated by the lack of published protocols and guides for the CR user. Most of our experiences were learned in practice, and we believed published guides would be useful for both existing users and future buyers. Careful attention to accuracy, histogram selection, positioning, collimation, and patient data input is critical to the success of CR. The persistent lack of a CR information system interface has led to occasional labeling errors and requires added vigilance on the part of both the technologist and the radiologist. Other important user problems we experienced with CR included the RVS. When images are reprinted from the RVS, they can be printed on 10 x 14 inch (26 x 36 cm) film only. Images that were acquired on the larger cassettes are printed on this smaller film, minified by one third from the original size. Minified images are unacceptable to our orthopedic colleagues. Networking problems have still not been resolved, and this resolution remains an important goal. The manipulation and transfer of images without loss of data were major reasons for our switch to electronic imaging and have still not been achieved. Unique image artifacts were encountered, as were new parameters to judge the quality of an image. The final issue is radiation dose. To assign dose reduction as an attribute of CR would be misleading. We have found that in many instances the reverse is true. Many examinations have required more radiation exposure than traditional film-screen techniques. At the least, to harvest the benefits of CR one must significantly alter technical exposure factors and understand how the system operates. The problems we encountered were not minor. Our technologists and radiologists and the Fuji personnel have put in many hours to implement and optimize our system. The image quality is superb, and we continue to work on the important networking and archival goals.

Aged↗

PubMed1990

Automated blood-sample handling in the clinical laboratory.

The only significant advances in blood-taking in 25 years have been the disposable needle and evacuated blood-drawing tube. With the exception of a few isolated barcode experiments, most sample-tracking is performed through handwritten or computer-printed labels. Attempts to reduce the hazards of centrifugation have resulted in air-tight lids or chambers, the use of which is time-consuming and cumbersome. Most commonly used clinical analyzers require serum or plasma, distributed into specialized containers, unique to that analyzer. Aliquots for different tests are prepared by handpouring or pipetting. Moderate to large clinical laboratories perform so many different tests that even multi-analyzers performing multiple analyses on a single sample may account for only a portion of all tests ordered for a patient. Thus several aliquots of each specimen are usually required. We have developed a proprietary serial centrifuge and blood-collection tube suitable for incorporation into an automated or robotic sample-handling system. The system we propose is (a) safe--avoids or prevents biological danger to the many "handlers" of blood; (b) small--minimizes the amount of sample taken and space required to adapt to the needs of satellite and mobile testing, and direct interfacing with analyzers; (c) serial--permits each sample to be treated according to its own "merits," optimizes throughput, and facilitates flexible automation; and (d) smart--ensures quality results through monitoring and intelligent control of patient identification, sample characteristics, and separation process.

Blood Specimen Collection↗

PubMed2005

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors.

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.

Amino Acid Sequence↗

PubMed1997

Rapid disruption of an astrocyte interaction with the extracellular matrix mediated by integrin alpha 6 beta 4 during focal cerebral ischemia/reperfusion.

BACKGROUND AND PURPOSE: Integrins participate in cerebral microvascular integrity and signaling during focal ischemia/ reperfusion. The integrin subunits alpha 1, alpha 6, and beta 1 are distributed identically on normal cerebral microvessels. Studies in epithelium indicate that integrin alpha 6 beta 4, which interacts with laminin-5 in the basal lamina/extracellular matrix, is unique. This study describes the exact location of alpha 6, beta 4, and alpha 6 beta 4 and that their responses in focal cerebral ischemia are relevant to astrocyte-matrix interactions. METHODS: The effect of middle cerebral artery occlusion and subsequent reperfusion on the microvascular expression of alpha 6 beta 4 and laminin-5 in regions of cellular injury (dUTP incorporation) was examined in 15 nonhuman primates. Well-characterized antibodies against human alpha 6, beta 4, alpha 6 beta 4, laminin-5 and laminin-1, endothelial CD31, and vascular markers were measured with computerized video imaging and laser confocal microscopy. RESULTS: Integrin alpha 6 beta 4 was localized on astrocytes where it connects with the extracellular matrix at the astrocyte-vessel interface. It represented 59.3 +/- 16.4% of alpha 6 antigen in cerebral microvessels < 100 microns in diameter. By 2 hours of ischemia, the significant reduction in alpha 6 expression (2P < .001) was accompanied by decreases in beta 4/laminin-5 (0.76 +/- 0.03 to 0.20 +/- 0.09; 2P = .001) and alpha 6 beta 4/laminin-5 (0.73 +/- 0.18 to 0.25 +/- 0.11; 2P = .001) in the region of dUTP incorporation. Parallel changes in laminin-5 and laminin-1 were less pronounced and coincided by 24 hours. CONCLUSIONS: This is the first description of a potential role of integrin alpha 6 beta 4 in the brain, where it mediates astrocyte-matrix interactions. The dramatic disappearance of alpha 6 beta 4 relative to its ligands reflects early loss of integrity between the astrocyte and the vessel wall in selected microvessels in response to ischemia.

Animals↗

PubMed2006

Entrez Gene: gene-centered information at NCBI.

Entrez Gene (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) is NCBI's database for gene-specific information. Entrez Gene includes records from genomes that have been completely sequenced, that have an active research community to contribute gene-specific information or that are scheduled for intense sequence analysis. The content of Entrez Gene represents the result of both curation and automated integration of data from NCBI's Reference Sequence project (RefSeq), from collaborating model organism databases and from other databases within NCBI. Records in Entrez Gene are assigned unique, stable and tracked integers as identifiers. The content (nomenclature, map location, gene products and their attributes, markers, phenotypes and links to citations, sequences, variation details, maps, expression, homologs, protein domains and external databases) is provided via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programing utilities (E-Utilities), and for bulk transfer by ftp.

Databases, Genetic↗

PubMedDate not supplied

Development of a highly detailed virtual model eye.

Using a three-dimensional modeling computer program, the authors created a three-dimensional, highly detailed computer-generated model of a normal eye to enhance the teaching of basic ophthalmic anatomy. The resulting model is a uniquely detailed virtual eye that can illustrate ocular anatomy clearly but realistically at many levels of magnification. Eye anatomy can be demonstrated from any viewpoint, with a choice of which structures are visible. The generation of video animations is also possible. The potential of this model for illustration of ocular disease will be the objective of further testing with medical students.

Computer Simulation↗

PubMed1995

New information technologies for healthcare in Aquitaine about the NursePad project.

The first results of a highly successful multidisciplinary NursePad Project in Aquitaine in France are to be presented as a scientific demonstration. A unique data collection system using electronic notepads has reduced the clerical workload of nurses with a further benefit of improved data quality. The system is well accepted by the health professionals using it because the software was designed by them for themselves. It addresses their problems. The developers are anxious to extend their findings into other domains of healthcare, both inside and outside the hospital.

France↗

PubMed2003

Characterization of silica-coated hematite and application to the formation of composite particles including egg yolk PC liposomes.

According to the method of Ohmori et al. (J. Colloid Interface Sci. 150 (1992) 594), a procedure is examined for the buildup of uniform silica layers on monodispersed hematite particles. It appears that the silica layer resulting is homogeneous and the layer thickness is controlled by the concentration of tetraethylorthosilicate (TEOS) in the medium. Further, egg PC liposomes, a typical biocolloid, are introduced onto the silica-coated hematite particle. The formation was proceeded by two types of processes: (1) heterocoagulation between the silica-coated hematite and egg PC liposomes by controlling the concentration of LaCl(3) in the medium, or (2) buildup using two proteins (lysozyme or cytochrome C) as binder molecules. These results were analyzed by zeta-potential measurements and a contact-type X-ray microscope, which is a unique technique for obtaining X-ray images of biological specimens in water with high resolution.

Cytochromes c↗

PubMed1996

A tRNA identity switch mediated by the binding interaction between a tRNA anticodon and the accessory domain of a class II aminoacyl-tRNA synthetase.

Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro. G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl-, glutaminyl-, and leucyl-tRNA synthetases. These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E. coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73. The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity.

Anticodon↗

PubMed2005

Automated on-line column-switching HPLC-MS/MS method with peak focusing for the determination of nine environmental phenols in urine.

We developed a method using isotope dilution on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination in urine of nine environmental phenolic compounds: Bisphenol A; 4-tert-octylphenol; o-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; benzophenone-3 (2-hydroxy-4-metoxybenzophenone); and triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether). A unique fully automated column-switching system, constructed using 1 autosampler, 2 HPLC pumps, and a 10-port switching valve, was designed to allow for concurrent SPE-HPLC operation with peak focusing. The phenols present in 100 microL of urine were retained and concentrated on a C18 reversed-phase size-exclusion SPE column. Then, the phenols were "back-eluted" from the SPE column and diluted through a mixing Tee before being separated from other urine matrix components using a pair of monolithic HPLC columns. The phenols were detected by negative ion-atmospheric pressure chemical ionization-MS/MS. The efficient preconcentration of the phenols by the SPE column, analyte peak focusing by the dilution, and minimal ion suppression in the LC/MS interface by the buffer-free mobile phases resulted in limits of detection as low as 0.1-0.4 ng/mL for most analytes. The method was validated on spiked pooled urine samples and on urine samples from 30 adults with no known occupational exposure to environmental phenols. The method can be used for quick and accurate analysis of large numbers of samples in epidemiologic studies for assessing the prevalence of human exposure to environmental phenols.

Chromatography, High Pressure Liquid↗

PubMed2006

Breaking the one antibody-one target axiom.

Studies at the interface of chemistry and biology have allowed us to develop an immunotherapeutic approach called chemically programmed antibodies (cpAbs), which combines the merits of traditional small-molecule drug design with immunotherapy. In this approach, a catalytic antibody catalyzes the covalent conjugation of a small molecule or peptide to the active site of the antibody, effectively recruiting the binding specificity of the conjugated molecule to the antibody. In essence, this technology provides the tools for breaking the "one antibody-one target axiom" of immunochemistry. Our studies in this area have focused on using the chemistry of the well studied aldolase catalytic antibodies of which mAb 38C2 is a member. Previously, we explored reversible assembly of cpAbs available through diketone chemistry. In this article, we explore a unique proadapter assembly strategy wherein an antibody 38C2-catalyzed transformation unveils a reactive tag that then reacts to form a stable covalent bond with the antibody. An integrin alpha(v)beta 3 antagonist was synthesized with the designed proadapter and studied using human breast cancer cell lines MDA-MB-231 and MDA-MB-435. We demonstrate that this approach allows for (i) the effective assembly of cpAbs in vitro and in vivo, (ii) selective retargeting of 38C2 to integrin alpha(v)beta 3 expressing breast cancer cell lines, (iii) intracellular delivery of cpAbs into cells, (iv) dramatically increased circulatory half-life, and (v) substantial enhancement of the therapeutic effect over the peptidomimetic itself in animal models of breast cancer metastasis. We believe that this technology possesses potential for the treatment and diagnosis of disease.

Animals↗

PubMed2002

Novel alpha7-like nicotinic acetylcholine receptor subunits in the nematode Caenorhabditis elegans.

We have used reverse-transcription-polymerase chain reaction (RT-PCR) and DNA sequencing techniques to confirm the transcription of seven (six alpha and one non-alpha) novel candidate nicotinic acetylcholine receptor (nAChR) subunit-encoding genes identified in the genome sequence of the nematode Caenorhabditis elegans. Compared to vertebrate nAChR subunits, they most closely resemble the homomer-forming, neuronal alpha7 subunit. Comparison of the predicted amino acid sequences of the new nAChR subunits with those described previously in C. elegans reveals five subunits (four alpha and one non-alpha) which resemble the DEG-3-like group of subunits. To date, this highly divergent nAChR subunit group is unique to C. elegans. ACR-22 is the first non-alpha member of the DEG-3-like group of subunits to be identified. Two new members of the related ACR-16-like nAChR group of subunits have also been shown to be transcribed, making the ACR-16-like subunit group the largest in C. elegans. Residues in the alpha subunit second transmembrane region (M2) which contribute to the channel lining show variations with implications for channel function. For example, in ACR-22, the highly conserved 0' lysine of M2 is replaced by histidine. Restrained molecular dynamics simulations have been used to generate molecular models of homo-pentameric M2 helix bundles for the novel subunits, enabling identification and display of pore-lining and protein interface residues. The number and diversity of genes encoding C. elegans nAChR subunits with similarities to the homomer-forming vertebrate alpha7 subunits and the identification of related non-alpha subunits, only found in C. elegans to date, suggest that at least some of these subunits may contribute to heteromers in vivo.

Amino Acid Sequence↗

PubMed1994

Differential expression of multiple cell-surface heparan sulfate proteoglycans during embryonic tooth development.

Heparan sulfate accumulates on cell surfaces and at cell-matrix interfaces, and functionally modulates several of the effector molecules that support the interactions, growth, and differentiation of developing tissues. Using heparin sulfate-specific monoclonal antibodies MAb, we obtained evidence that extracts from rodent embryos contain multiple forms of cell surface-associated heparan sulfate proteoglycan (PG). Taking tooth development in the mouse embryo as a model to further investigate the relevance of this PG redundancy and using MAb against heparan sulfate, antibodies specific for syndecan (syndecan-1) and fibroglycan (syndecan-2) (two distinct members of a larger family of cell-surface heparan sulfate PGs), and specific cDNA probes for these two cell-surface PGs, we obtained in situ evidence for regulated and differential expression of multiple cell-surface heparan sulfate PGs. The unique, distinctive, and coordinated changes in the expressions of these PGs during morphogenesis and differentiation of dental tissues suggest that the various cell-surface PGs are not truly redundant but play important, specific, and potentially complementary roles during embryonic development.

Animals↗

PubMed1998

Partonomies for interactive explorable 3D-models of anatomy.

We introduce a concept to model subtle part-whole-semantics for the use with interactive 3d-models of human anatomy. Similar to experiences with modeling partonomies for physical artifacts like machines or buildings we found one unique part-whole-relation to be insufficient to represent anatomical reality. This claim will be illustrated with anatomical examples. According to the requirements these examples demand, a semantic classification of part-whole-relations is introduced. Initial results in modeling anatomical partonomies for a 3d-visualization environment proved this approach to be an promising way to represent anatomy and to enable powerful complex inferences.

Anatomy↗

PubMed2006

Crystal structure of the catalytic domain of matrix metalloproteinase-1 in complex with the inhibitory domain of tissue inhibitor of metalloproteinase-1.

The mammalian collagenases are a subgroup of the matrix metalloproteinases (MMPs) that are uniquely able to cleave triple helical fibrillar collagens. Collagen breakdown is an essential part of extracellular matrix turnover in key physiological processes including morphogenesis and wound healing; however, unregulated collagenolysis is linked to important diseases such as arthritis and cancer. The tissue inhibitors of metalloproteinases (TIMPs) function in controlling the activity of MMPs, including collagenases. We report here the structure of a complex of the catalytic domain of fibroblast collagenase (MMP-1) with the N-terminal inhibitory domain of human TIMP-1 (N-TIMP-1) at 2.54 A resolution. Comparison with the previously reported structure of the TIMP-1/stromelysin-1 (MMP-3) complex shows that the mechanisms of inhibition of both MMPs are generally similar, yet there are significant differences in the protein-protein interfaces in the two complexes. Specifically, the loop between beta-strands A and B of TIMP-1 makes contact with MMP-3 but not with MMP-1, and there are marked differences in the roles of individual residues in the C-D connector of TIMP-1 in binding to the two MMPs. Structural rearrangements in the bound MMPs are also strikingly different. This is the first crystallographic structure that contains the truncated N-terminal domain of a TIMP, which shows only minor differences from the corresponding region of the full-length protein. Differences in the interactions in the two TIMP-1 complexes provide a structural explanation for the results of previous mutational studies and a basis for designing new N-TIMP-1 variants with restricted specificity.

Catalytic Domain↗

PubMed2004

FoldMiner and LOCK 2: protein structure comparison and motif discovery on the web.

The FoldMiner web server (http://foldminer.stanford.edu/) provides remote access to methods for protein structure alignment and unsupervised motif discovery. FoldMiner is unique among such algorithms in that it improves both the motif definition and the sensitivity of a structural similarity search by combining the search and motif discovery methods and using information from each process to enhance the other. In a typical run, a query structure is aligned to all structures in one of several databases of single domain targets in order to identify its structural neighbors and to discover a motif that is the basis for the similarity among the query and statistically significant targets. This process is fully automated, but options for manual refinement of the results are available as well. The server uses the Chime plugin and customized controls to allow for visualization of the motif and of structural superpositions. In addition, we provide an interface to the LOCK 2 algorithm for rapid alignments of a query structure to smaller numbers of user-specified targets.

Algorithms↗