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PubMed1980

Isolation and localization of DNA segments from specific human chromosomes.

Recombinant DNA techniques have been combined with somatic cell genetic methods to identify, isolate, and amplify fragments of human DNA localized at specific regions of human chromosome 11 selected as a model system. A library of genomic DNA segments has been constructed, in lambda Charon 4A bacteriophage, from the DNA of a somatic cell hybrid carrying a portion of human chromosome 11 on a Chinese hamster ovary cell background. Using a nucleic acid hybridization technique that distinguishes human and Chinese hamster interspersed, repetitive DNA, we have been able to distinguish recombinant phages carrying DNA segments of human origin from recombinant phages carrying DNA segments of Chinese hamster origin. We have isolated 50 human DNA segments thus far and have characterized 5 in detail. For each DNA segment characterized, a subsegment that carries no repetitive human DNA sequences has been identified. These segments have been used as hybridization probes in experiments that localize the DNA fragment on the chromosome. In each case an unequivocal chromosomal localization has been obtained with reference to a panel of hybrid cell clones each of which carries a deletion of a portion of the short arm of chromosome 11. At least one DNA segment has been identified which maps to each of the four regions on the short arm defined by the panel of hybrid cell clones used. The approaches described here appear to be general. They can be extended to produce a fine structure map of human chromosome 11 and other human chromosomes. This approach promises implications for human genetics generally, for the human genetic diseases, and possibly for understanding of gene regulation in normal and abnormal differentiation.

Animals↗
PubMed1989

Collagen fibrillogenesis in situ: fibril segments are intermediates in matrix assembly.

The assembly of discontinuous fibril segments and bundles was studied in 14-day chicken embryo tendons by using serial sections, transmission electron microscopy, and computer-assisted image reconstruction. Fibril segments were first found in extracytoplasmic channels, the sites of their polymerization; they also were found within fibril bundles. Single fibril segments were followed over their entire length in consecutive sections, and their lengths ranged from 7 to 15 microns. Structural differences in the ends of the fibril segments were identified, suggesting that the amino/carboxyl polarity of the fibril segment is reflected in its architecture. Our data indicate that fibril segments are precursors in collagen fibril formation, and we suggest that postdepositional fusion of fibril segments may be an important process in tendon development and growth.

Animals↗
PubMed1990

Influence of the hinge region on complement activation, C1q binding, and segmental flexibility in chimeric human immunoglobulins.

We have characterized a series of genetically engineered chimeric human IgG3 and IgG4 anti-dansyl (DNS) antibodies with identical antibody-combining sites but different hinge region amino acid compositions to determine how the hinge region influences Fab fragment segmental flexibility, C1q binding, and complement activation. Our data support the correlation between "upper hinge" length and Fab segmental flexibility; moreover, we confirm that a hinge region is essential for C1q binding and complement activation. However, the hinge length by itself is not sufficient for complement activity in IgG molecules. We have demonstrated that the IgG4 hinge, which imparts restricted segmental flexibility, reduces the ability of IgG3 molecules to activate complement. We also find that the IgG3 hinge region, which imparts greater segmental motion, is not sufficient to create complement activation activity in IgG4 anti-DNS antibodies. Finally, we conclude that (i) segmental motion is correlated with "upper hinge" length, (ii) hinge length and segmental flexibility is not enough to alter complement binding and activation, and (iii) segmental flexibility does not correlate with proficiency to activate the complement cascade.

Amino Acid Sequence↗
PubMed1993

Two nonpeptide tachykinin antagonists act through epitopes on corresponding segments of the NK1 and NK2 receptors.

The molecular mechanism of action for two chemically distinct and highly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was studied by development of a series of chimeric constructs between their respective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, substance P and neurokinin A, were not affected by exchanging almost the entire C-terminal half of the NK1 receptor with the corresponding segment of the NK2 receptor. In contrast, it was found that transfer from the NK2 to the NK1 receptor of a segment corresponding to transmembrane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the susceptibility for the nonpeptide antagonists. This chimeric exchange, corresponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK1 receptor--i.e., the Ki value for inhibition of binding of 125I-labeled substance P decreased from > 10,000 to 0.97 nM. At the same time the affinity for the NK1-selective compound CP-96,345 decreased > 30-fold. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [3H]SR-48,968, which did not bind to the NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor segment incorporated around transmembrane segments VI and VII, Kd = 1.5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act through epitopes located around transmembrane segment VI on their respective target receptors and that at least the nonconserved residues in these epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.

Animals↗
PubMed1994

Three-dimensional structure of the platelet integrin recognition segment of the fibrinogen gamma chain obtained by carrier protein-driven crystallization.

We have developed a method for crystallizing small functional protein segments so that their three-dimensional structure can be determined by x-ray diffraction analysis. This method consists of linking a small protein segment of unknown tertiary structure to either the amino or carboxyl terminus of a larger carrier protein of known tertiary structure. Crystallization of the small segment is then driven by crystallization of the carrier protein. Using this approach, we have obtained crystals of the human fibrinogen gamma-chain carboxyl-terminal segment linked to the carboxyl terminus of chicken egg white lysozyme. The three-dimensional structure of the carboxyl-terminal segment of the fibrinogen gamma chain was determined by x-ray diffraction analysis at a resolution of 2.4 A. This segment encompasses the recognition site for the integrin alpha IIb beta 3 receptor on activated platelets and for the clumping receptor on pathogenic staphylococci and also bears donor and acceptor sites for factor XIIIa-catalyzed crosslinking of fibrin. Therefore, the structural information derived from our analysis will provide a rational basis for the design of inhibitors of these important functions of fibrinogen. Moreover, carrier protein-driven crystallization will facilitate the determination of the three-dimensional structure of functional segments of other proteins that are, like fibrinogen, difficult to crystallize in toto.

Amino Acid Sequence↗
PubMed1996

Psychophysical evidence for fast region-based segmentation processes in motion and color.

Theories of image segmentation suggest that the human visual system may use two distinct processes to segregate figure from background: a local process that uses local feature contrasts to mark borders of coherent regions and a global process that groups similar features over a larger spatial scale. We performed psychophysical experiments to determine whether and to what extent the global similarity process contributes to image segmentation by motion and color. Our results show that for color, as well as for motion, segmentation occurs first by an integrative process on a coarse spatial scale, demonstrating that for both modalities the global process is faster than one based on local feature contrasts. Segmentation by motion builds up over time, whereas segmentation by color does not, indicating a fundamental difference between the modalities. Our data suggest that segmentation by motion proceeds first via a cooperative linking over space of local motion signals, generating almost immediate perceptual coherence even of physically incoherent signals. This global segmentation process occurs faster than the detection of absolute motion, providing further evidence for the existence of two motion processes with distinct dynamic properties.

Color Perception↗
PubMed1997

DNA sequence evidence for the segmental allotetraploid origin of maize.

It has long been suspected that maize is the product of an historical tetraploid event. Several observations support this possibility, including the fact that the maize genome contains duplicated chromosomal segments with colinear gene arrangements. Some of the genes from these duplicated segments have been sequenced. In this study, we examine the pattern of sequence divergence among 14 pairs of duplicated genes. We compare the pattern of divergence to patterns predicted by four models of the evolution of the maize genome-autotetraploidy, genomic allotetraploidy, segmental allotetraploidy, and multiple segmental duplications. Our analyses indicate that coalescent times for duplicated sequences fall into two distinct groups, corresponding to roughly 20.5 and 11.4 million years. This observation strongly discounts the possibility that the maize genome is the product of a genomic allotetraploid event, and it is also difficult to reconcile with either autotetraploidy or multiple independent segmental duplications. However, the presence of two (and only two) coalescent times is predicted by the segmental allotetraploid model. If the maize genome is the product of a segmental allotetraploid event, as these data suggest, then its two diploid progenitors diverged roughly 20.5 million years ago (Mya), and the allotetraploid event probably occurred approximately 11.4 Mya. Comparison of maize and sorghum sequences suggests that one of the two ancestral diploids shares a more recent common ancestor with sorghum than it does with the other ancestral diploid.

Journal Article↗
PubMed2000

Expression patterns of hairy, even-skipped, and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod.

There is an ongoing discussion on whether segmentation in different phyla has a common origin sharing a common genetic program. However, before comparing segmentation between phyla, it is necessary to identify the ancestral condition within each phylum. Even within the arthropods it is not clear which parts of the genetic network leading to segmentation are conserved in all groups. In this paper, we analyze the expression of three segmentation genes of the pair-rule class in the spider Cupiennius salei. Spiders are representatives of the Chelicerata, a monophyletic basic arthropod group. We find that in spider embryos, the orthologues for the Drosophila primary pair-rule genes hairy, even-skipped, and runt are expressed in stripes in the growth zone, where the segments are forming, suggesting a role for these genes in chelicerate segmentation. These data imply that the involvement of hairy, even-skipped, and runt in arthropod segmentation is an ancestral character for arthropods and is not restricted to a particular group of insects.

Animals↗
PubMed1995

A topological analysis of goldfish kainate receptors predicts three transmembrane segments.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.

Amino Acid Sequence↗
PubMed1996

Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4.

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-prekallikrein complex formation. Monoclonal antibody 13G11 binds to recombinant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epitope of 13G11 to the center portion of domain A4, positions 284-331. Direct binding studies of H-kininogen to various domain A4 constructs revealed that the distal H-kininogen binding portion is located on a segment of 48 residues, which overlaps the 13G11 epitope. Hence the tight interaction of H-kininogen and prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respectively, of the prekallikrein heavy chain. The isolated distal binding segment significantly prolongs the partial thromboplastin time of reconstituted Williams plasma thus stressing the critical role of the prekallikrein-H-kininogen complex formation in the initiation of the endogenous blood coagulation cascade.

Animals↗
PubMed1998

Regulatory segments of Ca2+/calmodulin-dependent protein kinases.

Catalytic cores of skeletal and smooth muscle myosin light chain kinases and Ca2+/calmodulin-dependent protein kinase II are regulated intrasterically by different regulatory segments containing autoinhibitory and calmodulin-binding sequences. The functional properties of these regulatory segments were examined in chimeric kinases containing either the catalytic core of skeletal muscle myosin light chain kinase or Ca2+/calmodulin-dependent protein kinase II with different regulatory segments. Recognition of protein substrates by the catalytic core of skeletal muscle myosin light chain kinase was altered with the regulatory segment of protein kinase II but not with smooth muscle myosin light chain kinase. Similarly, the catalytic properties of the protein kinase II were altered with regulatory segments from either myosin light chain kinase. All chimeric kinases were dependent on Ca2+/calmodulin for activity. The apparent Ca2+/calmodulin activation constant was similarly low with all chimeras containing the skeletal muscle catalytic core. The activation constant was greater with chimeric kinases containing the catalytic core of Ca2+/calmodulin-dependent protein kinase II with its endogenous or myosin light chain kinase regulatory segments. Thus, heterologous regulatory segments affect substrate recognition and kinase activity. Furthermore, the sensitivity to calmodulin activation is determined primarily by the respective catalytic cores, not the calmodulin-binding sequences.

Amino Acid Sequence↗
PubMed1998

Assessment of topogenic functions of anticipated transmembrane segments of human band 3.

Band 3 protein is a typical multispanning membrane protein whose membrane topology has been extensively studied from various protein chemical approaches. To clarify the membrane topogenesis of this multispanning protein on the endoplasmic reticulum, the topogenic functions of the anticipated transmembrane segments were individually assessed in an in vitro system using two series of model proteins in which each segment was placed in either a "stop-transfer" context or a "translocation initiation" context. They were expressed in a cell-free system containing rough microsomal membranes, and their topologies were evaluated by taking advantage of either sensitivity to protease or accessibility to N-glycosylation. We found that some segments seem to possess insufficient topogenic functions for membrane integration: the second transmembrane segment (TM2) is insufficient for the stop-transfer sequence, and TM3, TM5, and TM7 are not sufficient for the translocation initiation. In contrast to these phenomena, we herein demonstrate that TM2 shows an efficient stop-transfer function when it is near the preceding TM1 and suggest that TM3, TM5, and TM7 are followed by TM segments with a strong topogenic function to form Nexo/Ccyt topology, via which the preceding segments are integrated into the membrane. From these results, we propose that the interactions between the TMs should be operative during membrane integration, and that the segments with a weak topogenic function are given a transmembrane orientation by their following TMs.

Amino Acid Sequence↗
PubMed2002

The EIIIA segment of fibronectin is a ligand for integrins alpha 9beta 1 and alpha 4beta 1 providing a novel mechanism for regulating cell adhesion by alternative splicing.

Alternative splicing of the fibronectin gene transcript gives rise to forms that include the EIIIA (or ED-A) segment. EIIIA-containing fibronectins are prominently expressed during embryogenesis and wound healing and appear to mediate changes in cell adhesion and gene expression. Nonetheless, integrins that bind the EIIIA segment have not been identified. We previously mapped the epitope for two function-blocking monoclonal antibodies to the C-C' loop region of the EIIIA segment (Liao, Y.-F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884). The sequence of this epitope ((39)PEDGIHELFP(48)) resembles the sequence within tenascin-C to which the integrin alpha(9)beta(1) binds. We now report that either integrin alpha(9)beta(1) or alpha(4)beta(1) can mediate cell adhesion to the EIIIA segment. Moreover, this interaction is blocked both by epitope-mapped EIIIA antibodies as well as by the respective anti-integrins. Deletion mutants of the EIIIA segment that include the C-C' loop and flanking sequence bind cells expressing either alpha(9)beta(1) or alpha(4)beta(1). Adhesion of alpha(4)beta(1)-containing MOLT-3 cells to the EIIIA segment stimulates phosphorylation of p44/42 MAP kinase. Our observation that two integrins bind the EIIIA segment establishes a novel mechanism by which cell adhesion to fibronectin is regulated by alternative splicing.

Alternative Splicing↗
PubMed1999

Posterior segment inflammation in HLA-B27+ acute anterior uveitis: clinical characteristics.

PURPOSE: To examine clinical characteristics of patients who have posterior segment manifestations in HLA-B27-associated acute anterior uveitis. METHODS: Medical records of 114 HLA-B27-positive patients with acute anterior uveitis were reviewed in a retrospective fashion. Criteria for inclusion were the presence of acute anterior uveitis, a positive HLA-B27 antigen, and one of the following findings: vitreous cells >/=+2; cystoid macular edema; papillitis; vasculitis; or pars plana exudates. Twenty-four patients met the inclusion criteria. RESULTS: Posterior segment manifestations were found in 24 (21.05%) of 114 patients with HLA-B27+ acute anterior uveitis. Eighteen patients had diffuse vitritis (75%), seven had cystoid macular edema (29.1%), and two had papillitis (8.3%). Three patients had more than one finding. The prevalence of associated systemic disease (15 of 24 patients, 62.5%) and hypopyon (6 of 24 patients, 25%) in patients with posterior segment involvement was significantly higher than in the group of patients without posterior segment involvement (systemic disease, 33 of 90, 36.7%, p=0.04, exact chi-quare test; hypopyon, 4 of 90, 4.4%, p=0.006, exact chi-square test). The mean visual acuity at the last visit was 20/30 (range 20/20-20/100). Immunosuppressive therapy (other than corticosteroids) and surgical intervention were not required in the treatment of patients with posterior segment manifestations. CONCLUSION<?Pub Caret>: HLA-B27+ anterior uveitis is associated with posterior segment manifestations. Patients with posterior segment involvement have a significantly higher incidence of associated systemic diseases and hypopyon.

Acute Disease↗
PubMed2003

Pathophysiological characteristics of long- and short-segment Barrett's oesophagus.

BACKGROUND: Depending on the length of the segment of columnar epithelium in the distal oesophagus, Barrett's oesophagus can be divided into long-segment and short-segment Barrett's oesophagus. This article describes the pathophysiological characteristics of both forms of Barrett's oesophagus. METHODS: Review of the literature. RESULTS: Although there is some disagreement, ours and most other studies suggest that long-segment columnar-lined oesophagus is causally linked to chronic gastro-oesophageal reflux and that this from of Barrett's oesophagus is characterized by lower oesophageal sphincter tone, reduced oesophageal contractility and increased acid reflux. Short-segment Barrett's oesophagus is also associated with acid reflux, but the degree of oesophageal acid exposure and the level of other pathophysiological alterations seem to be lower. CONCLUSION: Pathophysiological abnormalities appear to be more prominent in long-segment Barrett's oesophagus than in short-segment Barrett's oesophagus.

Barrett Esophagus↗
PubMedDate not supplied

The relation between viable segments and left ventricular ejection fraction improvement.

For patients with coronary artery disease and left ventricular dysfunction who undergo revascularization, it is important to estimate the left ventricular ejection fraction (LVEF) improvement after revascularization, as this is a strong indicator of the long-term outcome. Identification of viable segments from echocardiography has been considered a predictive sign of LVEF improvement. However, a quantitative relation between segmental function recovery and global ejection fraction improvement has not been established. There is a clinical need to determine parameters that are predictive to LVEF improvement. A cylindrical left ventricular model is proposed to establish the relation between segmental myocardial function and LVEF based on a 12-segment echocardiograph model. Model results show that LVEF improvement is directly related to the contraction ratio in normal segments and a weighted sum of the number of viable segments that recover to normal or hypokinetic, which is equal to a weighted sum of the change in wall motion scores. This new combined parameter is a better predictor of the amount of LVEF improvement than the total number of viable segments or preoperative ejection fraction. The predictive value of the model was illustrated in a group of four patients with coronary artery disease who underwent revascularization.

Algorithms↗
PubMed1968

The small pyramidal neuron of the rat cerebral cortex. The axon hillock and initial segment.

The axon of the pyramidal neuron in the cerebral cortex arises either directly from the perikaryon or as a branch from a basal dendrite. When it arises from the perikaryon, an axon hillock is present. The hillock is a region in which there is a transition between the cytological features of the perikaryon and those of the initial segment of the axon. Thus, in the hillock there is a diminution in the number of ribosomes and a beginning of the fasciculation of microtubules that characterize the initial segment. Not all of the microtubules entering the hillock from the perikaryon continue into the initial segment. Distally, the axon hillock ends where the dense undercoating of the plasma membrane of the initial segment commences. Dense material also appears in the extracellular space surrounding the initial segment. The initial segment of the pyramidal cell axon contains a cisternal organelle consisting of stacks of flattened cisternae alternating with plates of dense granular material. These cisternal organelles resemble the spine apparatuses that occur in the dendritic spines of this same neuron. Axo-axonal synapses are formed between the initial segment and surrounding axon terminals. The axon terminals contain clear synaptic vesicles and, at the synaptic junctions, both synaptic complexes and puncta adhaerentia are present.

Animals↗
PubMed1973

Membrane characteristics and osmotic behavior of isolated rod outer segments.

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na(+) influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na(+) influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na(+) permeability constant of about 2.8 x 10(-6) cm/s and an osmotic permeability coefficient of greater than 2 x 10(-3) cm/s.

Animals↗