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PubMed1987

The structural basis of antigen-antibody recognition.

We have reviewed here the three-dimensional structure of an antigen-Fab complex as determined by X-ray crystallographic studies. The antigen is hen egg-white lysozyme (HEL), a protein whose three-dimensional structure and antigenic properties are well known. The Fab was prepared from a murine monoclonal anti-HEL antibody, IgGl,kappa, obtained by cell-hybridization techniques. The equilibrium association constant for the complex is 4.5 X 10(7) mol-1. The complex was crystallized and its three-dimensional structure was determined at 6-A and 2.8-A resolution. A three-dimensional model of the structure was built based on electron-density maps and the amino-acid sequence [determined from the nucleotide sequence of cDNA clones (M. Verhoeyen, C. Berek, J.M. Jarvis, G. Winter, in preparation)]. The three-dimensional structure of the complex shows that 17 antibody residues make close contacts (less than or equal to 4 A) with 16 antigen residues. Fifteen of the contacting antibody residues belong to the six complementarity-determining regions of the light chain (6 residues) and of the heavy chain (9 residues). The remaining two are located in regions of constant or nearly constant sequence ["framework" regions]. The 16 contacting lysozyme residues form a discontinuous, topographical determinant, since they are widely separated in the linear amino-acid sequence but are brought to relative spatial proximity by the three-dimensional folding of the polypeptide chain. The contacting surfaces are relatively flat, with protruding side chains of antigen and antibody penetrating each other over an area with maximum dimensions of 30 X 20 A. As in several other systems of protein-protein interactions, the contacts are chemically characterized as van der Waals interactions and hydrogen bonds. Detailed analysis of the interactions reveals that the antibody's recognition of the antigen is finely specific and is affected by antigenic variation (as observed in lysozymes from other avian species). The quaternary structure of the complexed Fab is elongated, with the axes of the variable (VH + VL) and constant (CH1 + CL) domains making an angle close to 180 degrees. Comparison of the three-dimensional structure of the complexed lysozyme with that of native lysozyme showed no significant conformational change at the current resolution (2.8 A). Comparison of the Fab moiety of the complex with other Fabs of known three-dimensional structure suggested that upon complexing no conformational change takes place in the tertiary structure of Fab either.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗

PubMed1986

Motion selectivity in macaque visual cortex. I. Mechanisms of direction and speed selectivity in extrastriate area MT.

Mechanisms of direction selectivity and speed selectivity were studied in single neurons of the middle temporal visual area (MT) of behaving macaque monkeys. Visual stimuli were presented in both smooth and stroboscopic motion within a neuron's receptive field as the monkey fixated a stationary point of light. Direction selectivity, speed selectivity, and the spontaneous discharge characteristics of MT neurons in behaving monkeys were similar to those reported in previous studies in anesthetized monkeys. Stroboscopic motion stimuli were sequences of flashes characterized by the spatial and temporal intervals between each flash. The spatial and temporal intervals were systematically varied so that suppressive and facilitatory interactions could be studied in both the preferred and null directions. Suppression and facilitation were measured by subtracting the peak discharge rate elicited by a single flash from the peak discharge rate elicited by a stroboscopic train of flashes. The dominant mechanism of direction selectivity in MT was a pronounced suppression of discharge for motion in the null direction which we interpreted as inhibition. The inhibition was sufficiently potent to abolish the responses to single flashed stimuli when they were embedded in a series of flashes in the null direction, and it frequently reduced the neuronal discharge to a level below the spontaneous firing rate. Facilitation in the preferred direction was a prominent feature of the responses of some, but not all, MT neurons. The peak discharge rate for stroboscopic motion in the preferred direction was more than twice the peak rate to a single flash for approximately 50% of the neurons in our sample. The direction selectivity of most MT neurons showed the effects of both inhibitory and facilitatory mechanisms, and it was not possible to segregate MT neurons into distinct groups on the basis of these measures. Suppressive mechanisms contributed to speed tuning as well as direction tuning. The low-speed cutoff for motion in the preferred direction resulted from suppression in 82% of the neurons tested. The high-speed cutoff resulted from suppression in 32% of the neurons tested. The latter mechanism appeared to be distinct from the inhibitory mechanism which acted in the null direction in that large spatial intervals were required for its activation.

Animals↗

PubMed1997

Myogenin can substitute for Myf5 in promoting myogenesis but less efficiently.

The myogenic basic Helix-Loop-Helix transcription factors, including Myf5, MyoD, myogenin (myg) and MRF4, play important roles in skeletal muscle development. The phenotypes of mutant mice deficient in either gene are different, suggesting that each gene may have a unique function in vivo. We previously showed that targeting myogenin into the Myf5 locus (Myf5(myg-ki)) rescued the rib cage truncation in the Myf5-null mutant, hence demonstrating functional redundancy between Myf5 and myogenin in skeletal morphogenesis. Here we present the results of crossing myogenin knock-in (myg-ki) mice with either MyoD-null or myogenin-null mutants. The Myf5(myg-ki) allele rescued early myogenesis, but Myf5(myg-ki/myg-ki);MyoD(-/-) mutant mice died immediately after birth owing to reduced muscle formation. Therefore, myogenin, expressed from the Myf5 locus, is not able to completely replace the function of Myf5 in muscle development although it is capable of determining and/or maintaining myogenic lineage. Myf5(myg-ki/myg-ki);myg(-/-) mutant mice displayed the same phenotype as myg(-/-) mutants. This indicates that the earlier expression of myogenin cannot promote myogenic terminal differentiation, which is normally initiated by the endogenous myogenin. Thus, our results are consistent with the notion that Myf5 and myogenin are functionally interchangeable in determining myogenic lineage and assuring normal rib formation. Our experiment revealed, however, that some aspects of myogenesis may be unique to a given myogenic factor and are due to either different regulatory sequences that control their temporal and spatial expression or different functional protein domains.

Alleles↗

PubMed2001

High resolution CISS imaging of the spine.

Spatial resolution remains one of the major problems and goals in spinal imaging. The high spatial resolution afforded by a novel sequence, constructive interference in steady state (CISS), provides a further refinement to MRI, the modality of choice in the investigation of suspected intraspinal pathology. Both complex and subtle abnormalities are more fully elucidated using CISS. It is now used in our institution as an adjunct to conventional imaging sequences in the diagnostic evaluation of complex intraspinal pathology. The anatomical information provided by CISS is of particular value in planning surgical interventions, most notably in the management of intraaxial and extraaxial cystic abnormalities, dysraphic malformations and disturbances of cerebrospinal fluid circulation, including post-traumatic and post-surgical scarring.

Arachnoiditis↗

PubMed1985

[Flow analysis by phase modulation of NMR signals].

Time modulated field-gradient-sequences able to selectively phase encode spatial location or flow velocity in NMR signals are described. The specific phase modulation discriminates between stationary and mobile protons. The method is presented in the one dimensional case and has the following advantages: it is sensitive to flow direction and unaffected by the relaxation times.

Magnetic Resonance Spectroscopy↗

PubMed2000

Muscarinic receptor activation promotes the membrane association of tubulin for the regulation of Gq-mediated phospholipase Cbeta(1) signaling.

The microtubule protein tubulin regulates adenylyl cyclase and phospholipase Cbeta(1) (PLCbeta(1)) signaling via transactivation of the G-protein subunits Galphas, Galphai1, and Galphaq. Because most tubulin is not membrane associated, this study investigates whether tubulin translocates to the membrane in response to an agonist so that it might regulate G-protein signaling. This was studied in SK-N-SH neuroblastoma cells, which possess a muscarinic receptor-regulated PLCbeta(1)-signaling pathway. Tubulin, at nanomolar concentrations, transactivated Galphaq by the direct transfer of a GTP analog and potentiated carbachol-activated PLCbeta(1). A specific and time-dependent association of tubulin with plasma membranes was observed when SK-N-SH cells were treated with carbachol. The same phenomenon was observed with membranes from Sf9 cells, expressing a recombinant PLCbeta(1) cascade. The time course of this event was concordant both with transactivation of Galphaq by the direct transfer of [(32)P]P(3)(4-azidoanilido)-P(1)-5'-GTP from tubulin as well as with the activation of PLCbeta(1). In SK-N-SH cells, carbachol induced a rapid and transient translocation of tubulin to the plasma membrane, microtubule reorganization, and a change in cell shape as demonstrated by confocal immunofluorescence microscopy. These observations presented a spatial and temporal resolution of the sequence of events underlying receptor-evoked involvement of tubulin in G-protein-mediated signaling. It is suggested that G-protein-coupled receptors might modulate cytoskeletal dynamics, intracellular traffic, and cellular architecture.

Animals↗

PubMedDate not supplied

Three-dimensional geometrical models of the inguinal region. Towards a new stereology.

In this work we studied the inguinal-abdominal region and the inguinal canal using three-dimensional geometrical models. We built the models through computer aided geometric modeling techniques on the basis of observations during real dissections, operations and diagnostic medical imaging. The obtained models show in a complete modular synthesis and with a schematic iconology the structural organization of the anatomical districts in a logic sequence of layers and topographic and spatial relationships among its components. The models represent an amazing support to anatomy and clinical anatomy for teaching and research purposes on organogenesis, surgery and diagnosis.

Abdominal Muscles↗

PubMed1992

Cardiac magnetic resonance spectroscopy.

Magnetic resonance spectroscopy provides information about metabolic processes in living tissues. The resulting information may be more sensitive and specific than that obtained using techniques that rely on functional or structural measurements. Although the preponderance of applications to date have been in physiologic investigations in animals, clinical applications are emerging. Reports in the past two years have appeared evaluating the clinical use of phosphorus spectroscopy to detect ischemic heart disease, cardiomyopathy, and cardiac transplant rejection. Active research using nuclei other than phosphorus for spectroscopy will expand the potential clinical applications. Technical developments, including improved surface coil design, wider use of high-field magnets and new pulse sequences, will allow improved sensitivity and spatial localization in the future.

Animals↗

PubMedDate not supplied

Three-dimensional geometrical models of the liver.

In this work we used a virtual approach to study the human liver by three-dimensional geometrical models. We built the models through computer aided geometric modelling techniques starting from pictures taken during both real dissections and diagnostic medical imaging. The results show in a complete modular synthesis and with a schematic iconology the structural organization of this organ in a logic sequence of layers and topographic and spatial relationship among its components. This approach represents an amazing support to clinical anatomy for teaching and research.

Computer Simulation↗

PubMed1998

Cooperating morphogens control hoxd gene expression in the developing vertebrate limb.

A mechanism was recently proposed which controls the expression of Hoxa genes in the developing vertebrate limb. This is a three-dimensional diffusion model of a morphogen Ma secreted at the apical ectodermal ridge. The morphogen Ma is at the same time degraded by first order chemical kinetics. The emerging diffusion gradient, supplemented with a natural sequence of thresholds, can explain the spatial and temporal collinearities of the Hoxa gene expression domains. In the present model it is assumed that a second diffusing morphogen Mz is produced at the zone of polarizing activity. A concentration superposition of morphogens Ma and Mz can then explain the observed pattern of expression of Hoxd-10, 11, 12, and 13 during normal limb development (spatial and temporal collinearity). The associated thresholds for gene expression increase sequentially from Hoxd-10 to Hoxd-13 (threshold collinearity). The observed deformations or deletions of these expression domains in manipulated limbs are also explained by the model. Additional model predictions are proposed to test the validity of the model. In patterning two mechanisms are mainly considered as responsible for signalling: (i) a gradient of a long-range morphogen created by passive diffusion; and (ii) a short-range inducer initiating a cascade of consecutive signals. A time-test is proposed which distinguishes the modes of specific gene expressions for these distinct signalling mechanisms.

Animals↗

PubMedDate not supplied

The role of nucleotide sequences in splice site selection in eukaryotic pre-messenger RNA.

Alternative splicing of eukaryotic messenger RNA precursors is now known to be of widespread importance in generating multiple transcripts from a single gene. This phenomenon has emphasized the problem of the way in which splice sites are selected; recent studies have discussed the role of secondary structure or affinity and spatial relationships in this selection. Splice site sequences vary widely, although a loose consensus has been derived for the 9 bases around the 5' splice site and for a longer region around the 3' splice site. Mutagenesis experiments have defined the sequences essential for a potential 5' splice site, but, except for some experiments with the E1a gene of adenovirus, these experiments have not examined 5' splice site sequences for features responsible for site preference where alternative splicing sites exist. Such tests require a choice of site: an appropriate reference site and a constant position at which test sites are introduced. We have begun a series of experiments designed to show whether splice site sequences can be ranked in a hierarchy of preferential use. Here we show that the archetypal consensus sequence is used efficiently, and characterize the cryptic sites of beta-globin: sequences alone can explain why these sites are not normally used. We also show with the E1a gene of adenovirus, a simple example of alternative splicing, that one of the two 5' splice sites used by this gene is intrinsically stronger. We also demonstrate that tandem repeats and secondary structure influence the choice of sites in vivo. We discuss the mechanism of splice site selection.

Adenovirus Early Proteins↗

PubMed2000

Genetic diversity of rice tungro spherical virus in tungro-endemic provinces of the Philippines and Indonesia.

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed.

Animals↗

PubMed2000

Helical representation of atrial reentry: a teaching aid for electrophysiology.

Cardiac clinical electrophysiology is difficult to teach because it requires mental integration of complex information on cardiac activation, which includes tridimensional spatial orientation of the cardiac structures involved and variables such as refractory period, conduction velocity, and cycle length. Commonly used representations of cardiac arrhythmias include snapshot sequences or ladder diagrams. The former lack time dimension, and the latter lack spatial dimension. We propose a schematic tridimensional representation of reentry as a helicoidal shape that is the result of adding a time dimension to a circular representation. This presentation, which also can be called a "loop diagram," allows better integration of spatial phenomena with recorded electrogram sequences and can help the teaching of basic clinical electrophysiology.

Electrophysiology↗

PubMed2005

Intramural activation and repolarization sequences in canine ventricles. Experimental and simulation studies.

BACKGROUND: There are no published data showing the three-dimensional sequence of repolarization and the associated potential fields in the ventricles. Knowledge of the sequence of repolarization has medical relevance because high spatial dispersion of recovery times and action potential durations favors cardiac arrhythmias. In this study we describe measured and simulated 3-D excitation and recovery sequences and activation-recovery intervals (ARIs) (measured) or action potential durations (APDs) (simulated) in the ventricular walls. METHODS: We recorded from 600 to 1400 unipolar electrograms from canine ventricular walls during atrial and ventricular pacing at 350-450 ms cycle length. Measured excitation and recovery times and ARIs were displayed as 2-D maps in transmural planes or 3-D maps in the volume explored, using specially developed software. Excitation and recovery sequences and APD distributions were also simulated in parallelepipedal slabs using anisotropic monodomain or bidomain models based on the Lou-Rudy version 1 model with homogeneous membrane properties. RESULTS: Simulations showed that in the presence of homogeneous membrane properties, the sequence of repolarization was similar but not identical to the excitation sequence. In a transmural plane perpendicular to epicardial fiber direction, both activation and recovery pathways starting from an epicardial pacing site returned toward the epicardium at a few cm distance from the pacing site. However, APDs were not constant, but had a dispersion of approximately 14 ms in the simulated domain. The maximum APD value was near the pacing site and two minima appeared along a line perpendicular to fiber directions, passing through the pacing site. Electrical measurements in dog ventricles showed that, for short cycle lengths, both excitation and recovery pathways, starting from an epicardial pacing site, returned toward the epicardium. For slower pacing rates, pathways of recovery departed from the pathway of excitation. Highest ARI values were observed near the pacing site in part of the experiments. In addition, maps of activation-recovery intervals showed mid-myocardial clusters with activation-recovery intervals that were slightly longer than ARIs closer to the epi- or endocardium, suggesting the presence of M cells in those areas. Transmural dispersion of measured ARIs was on the order of 20-25 ms. Potential distributions during recovery were less affected by myocardial anisotropy than were excitation potentials.

Action Potentials↗

PubMed2005

Selective targeting of indel-inferred differences in spatial structures of highly homologous proteins.

Recent findings have shown that the protein elongation factor-1alpha (EF-1alpha) from the eukaryotic pathogen Leishmania donovani possesses virulence properties. This was unexpected, since it has greater than 80% sequence identity with its human homologue. Given that EF-1alpha is essential for cell survival, in principle, it can be considered an attractive drug target. However, the challenge is to be able to selectively target the protein so as not to affect function of the human homologue. While a limited number of discrete differences were scattered throughout the sequence, most of the difference between these 2 homologues could be attributed to a 12-amino acid insert present in human EF-1alpha and absent from the leishmania sequence. In the present study, we modeled the spatial differences in structures of human and L. donovani EF-1alpha's inferred by this insertion-deletion (or "indel"). The protein models were used to develop antibodies directed specifically toward the deletion region of the pathogen protein. The strategy described allowed successful selective targeting of this putative leishmania virulence factor while avoiding recognition of the highly similar human EF-1alpha homologue. These findings may establish a new strategy for the development of antagonists directed against certain pathogenic targets having close human homologues.

Amino Acid Sequence↗

PubMed1993

Quantitative assessment of the microstructure of rat behavior: I, f(d), the extension of the scaling hypothesis.

Previous studies demonstrated that drug effects on the movement sequences of rats in unconditioned motor activity paradigms can be quantified by scaling measures that describe the average relationship between a variable of interest and an experimental parameter. However, rats engage in a wide variety of geometrically distinct movements that can be influenced differentially by drugs. In this investigation, the extended scaling approach is presented to capture quantitatively the relative contributions of geometrically distinct movement sequences to the overall path structure. The calculation of the spectrum of local spatial scaling exponents, f(d), is based on ensemble methods used in statistical physics. Results of the f(d) analysis confirm that the amount of motor activity is not correlated with the geometrical structure of movement sequences. Changes in the average spatial scaling exponent, d, correspond to shifting the entire f(d) function, and indicate overall changes in path structure. With the extended scaling approach, straight movement sequences are assessed independently from highly circumscribed movements. Thus, the f(d) function identifies drug effects on particular ranges of movement sequences as defined by the geometrical structure of movements. More generally, the f(d) function quantifies the relationship between microscopically recorded variables, in this paradigm consecutive (x,y) locations, and the macroscopic behavioral patterns that constitute the animal's response topography.

Animals↗

PubMed2026

Single-cell and spatial transcriptomics define a progenitor subpopulation and fibroinflammatory niche at the leading edge of parathyroid carcinoma.

Parathyroid carcinoma (PC) is a rare but clinically aggressive endocrine malignancy with limited treatment options and a poorly defined tumor microenvironment (TME). To elucidate its cellular heterogeneity and spatial architecture, we integrated single-cell and spatial transcriptomic profiling with whole-exome sequencing and multiplex immunohistochemistry on eight parathyroid neoplasm specimens, including PC, parathyroid adenoma, and atypical parathyroid tumor. We identified a distinct progenitor-like endocrine subpopulation (Ca-1) enriched in CDC73-mutant PC, exhibiting stem-like properties, elevated cell cycle activity, and pronounced genomic instability. Spatial mapping revealed that Ca-1 cells preferentially localize at the leading edge, forming a fibroinflammatory niche characterized by the enrichment of inflammatory cancer-associated fibroblasts (iCAFs) and SPP1+ macrophages. Within this niche, the dipeptidyl peptidase 4 (DPP4) is selectively expressed in Ca-1 cells and iCAFs, implicating a potential paracrine axis driving stromal remodeling and immunosuppression. These findings suggest that a spatially organized ecosystem may promote PC progression through TME remodeling and highlight the DPP4-CXCL2 axis as a candidate pathway for future investigation in aggressive parathyroid neoplasms.

Humans↗

PubMed2003

Structural localization of disease-associated sequence variations in the NACHT and LRR domains of PYPAF1 and NOD2.

Several autoinflammatory diseases with distinct clinical manifestations have been associated with sequence variations in the gene products PYPAF1/CIAS1 and NOD2/CARD15. Both proteins belong to the PYD/CARD-containing family of apoptosis regulators and activators of pro-inflammatory caspases. To gain insight into the dysfunctional role of sequence alterations, we assembled a structure-based multiple sequence alignment of family members and related proteins. This allowed us to analyze the putative effect of the alterations on the function of nucleotide-binding (NACHT) and leucine-rich repeat (LRR) domains shared by the family members. In support of this analysis, we carefully selected template structures for the NACHT and LRR domains and mapped the genetic variations onto 3D domain models. Additionally, we propose a model of the NACHT and LRR domain complex. Our study revealed that many of the disease-associated sequence variants are located close to highly conserved sequence regions of functional relevance and are spatially adjacent in the predicted 3D structure. The implications on the domain functions such as NTP-hydrolysis or oligomerization are discussed.

Amino Acid Sequence↗