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PubMed1975

Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.

The three-dimensional structure of carbonic anhydrase B (EC 4,2,1,1; carbonate hydro-lyase) from human erythrocytes has been determined to high resolution. Parallel and antiparallel pleated sheet makes up the predominant secondary structure of the enzyme. The tertiary structure is unique for its folding and is very similar to the structure is unique for its folding and is very similar to the structure of the isoenzyme, human erythrocyte carbonic anhydrase C. The essential metal ion, zinc, is firmly bound to the enzyme through three histidyl ligands and located at the bottom of a 12-A deep conical cavity. The zinc ligands are involved in a number of hydrogen bond formations with residues in the immediate vicinity of the active site cavity. Some of the similarities and differences in the sidechain orientation and active site topography of the two isoenzymes are also discussed.

Binding Sites↗
PubMed1975

Model of protein folding: inclusion of short-, medium-, and long-range interactions.

A hypothesis for protein folding is proposed, in which the native structure is formed by a three-step mechanism: (A) formation of ordered backbone structures by short-range interactions, (B) formation of small contact regions by medium-range interactions, and (C) association of the small contact regions into the native structure by long-range interactions. Empirical interaction parameters (free energy of formation of a contact) between amino-acid residues were evaluated from the frequency of contacts in the x-ray structures of native proteins. On the basis of this mechanism, a Monte Carlo simulation of protein folding (with an accompanying decrease in the total contact free energy) was carried out for bovine pancreatic trypsin inhibitor. The predicted three-dimensional structure is in fairly good agreement with the experimental one.

Mathematics↗
PubMed1976

Three dimensional structure of erabutoxin b neurotoxic protein: inhibitor of acetylcholine receptor.

The three-dimensional structure of erabutoxin b, a neurotoxin in the venom of the sea snake Laticauda semifasciata, has been determined from a 2.75 A resolution electron density map. Erabutoxin b is one of a family of snake venom neurotoxins, all low-molecular-weight proteins, which block neuromuscular transmission at the postsynaptic membrane. They specifically inhibit the acetylcholine receptor. The molecular shape is that of a shallow elongated saucer with a footed stand formed by the six-membered ring at the COOH-terminal end. The central core of the molecule is an assembly of four disulfide bridges. Three long chain loops emerge as broad fronds from the core region. Approximately 40% of the main chain is organized into a twisted antiparallel beta-pleated sheet of five short strands. In 28 snake venom neurotoxins of established sequence which inhibit the acetylcholine receptor, the four disulfide bridges and seven other residues remain invariant. Three substitution positions conserve residue type. In one wing of the molecule, there is a broad shallow depression which may characterize the reactive site. It is populated by the sevent invariant residues and two of the three type conserved residues. This region is "anchored" on the undersurface of the molecule by the hydroxyl group of Ser-9, the remaining conservatively substituted residue.

Erabutoxins↗
PubMed1977

Membrane-controlled depletion of complement activity by spin-label-specific IgM.

Complement depletion mediated by high molecular weight (IgM) rabbit antibodies specifically bound to spin-label lipid haptens dispersed in model membranes is controlled by various physical attributes of those membranes other than the total number of exposed determinants that they provide. Carrier lipids used at 32 degrees were (i) a "fluid" phosphatidylcholine (PC), (ii) a "solid" PC, and (iii) a cholesterol/PC mixture. The concentration of hapten in the plane of the membranes (two-dimensional concentration) was varied while the overall hapten molarity (three-dimensional concentration) was kept constant. Both specific binding and the efficiency of depletion by IgM are markedly enhanced by systematically decreasing the average distance between haptens (infinity --> 26 A). Heterogeneous distribution was found to be more favorable than a random homogeneous distribution of the same number of haptens in the same total quantity of lipids. IgM efficiency is also markedly increased by the inclusion of cholesterol in PC membranes, an effect thought to result from enhanced projection of the determinant from the surface of the membrane and hence increased accessibility to the antibody-binding site. Furthermore, the efficiency of IgM was increased by using haptens dispersed in fluid rather than in solid PC membranes. The results are consistent with the hypothesis that IgM molecules must be bound to a critical multiple of antigenic determinants at a membrane surface in order to induce complement-mediated attack and that subtle variation of the physical state of membrane antigens can be the crucial factor in determining the outcome of this type of efferent immune response.

Animals↗
PubMed2025

Knowledge-enhanced protein subcellular localization prediction from 3D fluorescence microscope images.

MOTIVATION: Pinpointing the subcellular location of proteins is essential for studying protein function and related diseases. Advances in spatial proteomics have shown that automatic recognition of protein subcellular localization from images could highly facilitate protein translocation analysis and biomarker discovery, but existing machine-learning works have been mostly limited to processing 2D images. By contrast, 3D images have higher spatial resolution and allow researchers to observe cellular structures in their natural context, but currently, there are only a few studies of 3D image processing for protein distribution analysis due to the lack of data and complexity of modeling. RESULTS: We developed a knowledge-enhanced protein subcellular localization model, KE3DLoc, which could recognize distribution patterns in 3D fluorescence microscope images using deep learning methods. The model designs an image feature extraction module that incorporates information from 3D and 2D projected cells and implements asymmetric loss and confidence weights to address data imbalance and weak cell annotation issues. Besides, considering that the biological knowledge in the Gene Ontology (GO) database can provide valuable support for protein location understanding, the KE3DLoc model incorporates a novel knowledge enhancement module that optimizes the protein representation by related knowledge graphs derived from the GO. Since the image module and the knowledge module calculate features from different levels, KE3DLoc designs protein ID aggregation to enhance the consistency of protein features across different cells. Experimental results on three public datasets have demonstrated that the KE3DLoc significantly outperforms existing methods and provides valuable insights for spatial proteomics research. AVAILABILITY AND IMPLEMENTATION: All datasets and codes used in this study are available at GitHub: https://github.com/PRBioimages/KE3DLoc.

Microscopy, Fluorescence↗
PubMed2025

TRB proteins in moss reveal their evolutionarily conserved roles in plant development and telomere maintenance.

Telomere repeat binding (TRB) proteins are plant-specific proteins with a unique domain structure distinct from telomerebinding proteins in animals and yeast. While extensively studied in seed plants, their role in early-diverging plant lineages remains largely unexplored. Here, we investigate TRB proteins in a model moss, Physcomitrium patens, to assess their evolutionary conservation and functional significance. Functional analysis using single knockout mutants revealed that individual PpTRB genes are essential for normal development, with mutants exhibiting defects in the two-dimensional (protonemal) stage, and more prominently, in the formation of three-dimensional (gametophore) structures. Some double mutants displayed telomere shortening, a phenotype also observed in TRB-deficient seed plants, indicating a conserved role for TRBs in telomere maintenance. Transcriptome profiling of TRB mutants revealed altered expression of genes associated with transcriptional regulation and stimulus response in protonema. Subcellular localization studies across various plant cell types confirmed that PpTRBs, like their seed plant counterparts, localize prevalently to the plant nucleus and mutually interact. In bryophytes, TRBs form a monophyletic group that mirrors the species phylogeny, whereas in seed plants, TRBs have diversified into two distinct monophyletic groups. Our findings provide the first comprehensive characterization of TRB proteins in non-vascular plants and demonstrate their conserved roles in telomere maintenance, with additional implications for plant development and gene regulation across land plant lineages.

Bryopsida↗
PubMed1978

Study of pole assembly in Bacillus subtilis by computer reconstruction of septal growth zones seen in central, longitudinal thin sections of cells.

The septal growth of Bacillus subtilis 168/s has been studied by making a number of observations from thin sections of cells from exponentially growing cultures. The process was initiated by the formation of a new cross wall under a preexisting layer of cylindrical wall. An annular notch appeared to cut through the overlying wall and presumably allowed the cross wall to split into two layers of peripheral wall. During this initial notching process, two raised bands of wall material were produced which resembled those previously observed in morphological studies of Streptococcus faecalis. Through an improved fixation technique, it was possible to preserve the bands seen in B. subtilis to the extent that they were used as markers to study the subsequent stages of septal growth. These stages included (i) the continued displacement of the two bands from the cross wall (as the two nascent polar surfaces enlarged and as the diameter of the cross wall decreased), (ii) the closure of the cross wall, and (iii) the final severance of the common cross wall connection between two completed poles. To study this process in a more quantitative manner, three-dimensional reconstructions of the envelope observed between pairs of the raised bands were made from axial thin sections of cells. The process of reconstruction was based on a technique by which x, y coordinates were taken from thin sections and were rotated around the cell's central axis. These reconstructions were used to estimate the surface area or volume of the reconstructed zones or their parts. A round of septal growth was then simulated by arranging 118 reconstructions in order of increasing surface area or volume. The topology of the process was studied by noting how various measurements of septal thickness, length, surface area, and volume varied as a function of increasing septal zone size. This analysis was based on several assumptions, of which three of the most important are: (i) the bands produced by the initial notching process are markers which separate septal from cylindrical wall growth; (ii) a septal zone observed between pairs of bands is made up of two nascent poles and a single cross wall; and (iii) as septal zones develop in terms of relative age they increase in size (volume or surface area) or amount of wall. The data suggested that the S. faecalis model of surface growth (in which polar growth occurs through a regulated constrictive separation and expansion of a cross wall) also seems applicable to the pattern of septal growth observed here for B. subtilis. This was indicated from measurements which showed that increases in the size of nascent polar surfaces were correlated with decreases in cross wall diameter. An explanation of these observations may be that decreases in cross wall diameter were due to a progressive splitting of the cross wall that removed surface from the outer circumference of the cross wall and converted it into new polar surface. Calculations further suggested that if the poles of B. subtilis were made by this model a sizeable and variable increase in surface area of the cross wall would also be required to convert these separating cross wall layers into two curved polar structures. Measurements of wall thickness taken from various locations within septal zones indicated that while the thickness of the polar wall of B. subtilis was constant over its surface, the width of the cross wall varied considerably during a round of synthesis. Again, one of the simplest explanations compatible with these observations and those previously made in S. faecalis is that the B. subtilis cross wall is brought to a constant thickness (possibly by remodeling or precursor addition) before or during separation. Although most observations made from the reconstruction of the septal zones of B. subtilis may fit the S. faecalis model of surface growth, differences in the pattern of septal growth were seen when the two organisms were compared. These have been discussed in terms of differences in the regulation of their respective septal growth sites and basic mechanisms of wall assembly and modification.

Bacillus subtilis↗
PubMed1976

The biological origin of antibody diversity.

Antibody diversity has a compelling fascination for many scientists and over the years speculations have sometimes seemed more numerous than facts. Now the structural basis of antibody specificity is well defined. Amino acid sequences and recently three-dimensional structures of various immunoglobulins provide the most solid basis for discussing the origin of diversity. The novel pattern of variable (V) and Constant (C) regions of amino acid sequence has been resolved further to show the functional pattern of variability. Inheritance of separate V and C genes is accepted, but attempts to define more than one gene coding for each V region are considered here to be unnecessary. The pattern of variability is still best understood in terms of mutation and the presence or absence of various selective pressures. The major area of debate still hinges around the extent to which mutation and selection operate during evolution or somatically. Sequence data have now been generally interpreted to require multiple V genes carried in the germ line. A few individual VH genes have been mapped in close linkage to CH genes in the mouse. The apparent existence of three VH alleles in rabbits was a strong argument against multiple V genes. Now the three phenotypes have been shown to be due to alleles controlling the expression of three sets of VH genes all present on the same chromosome. That V-gene expression requires rejoining of V and C genes at the DNA level is now almost certain. Models for the joining process can draw on the precedents of transposable genetic elements, which are widespread in Nature. The total extent of antibody diversity remains a philosophical point. Estimates of the number of antibody molecules required for observed diversity are reduced by two recently documented proposals. Each antibody combining site apparently has many (estimated at 100) different specificities and most combinations of VH and VL regions probably form a viable site. A given combining site can be defined by its pattern of shared specificities. Several specific antibody repertoires have been measured and the size in each case is consistent with the stringency with which the specificity is selected. Repertoire size appears to be under genetic control, but there are problems in viewing the genotype through the veil of clonal selection. Molecular hybridization has been used recently in an attempt to count V and C genes directly. C genes are seen in DNA having nonreiterated sequences, as formal genetics predicts. Each V-region probe hybridizes at a similar rate to C-region probes. Interpretation of this result depends on the extent to which one V-region probe will reveal nonhomologous V genes. Previous estimates that many cross-hybridizing genes should have been seen if present are possibly exaggerated. It is argued here that the data are compatible with a germ-line gene for each probe studied. Maximum estimates for the number of germ-line genes are sufficient to account for antibody diversity...

Amino Acid Sequence↗
PubMed1977

The use of photogrammetry in tissue compensator design. Part I: photogrammetric determination of patient topography.

The surface topography of a patient can be determined by photogrammetry before beginning radiotherapy. The source light of the therapy unit or simulator is used to project a grid pattern onto the patient, and this is then photographed together with control points consisting of miniature light bulbs mounted on a frame suspended from the wedge slot of the therapy machine. When the photograph is projected onto a graphics terminal for data entry into a computer, the three-dimensional topography of the patient's surface can be reconstructed as a two-dimensional matrix of discrete points. A computer algorithm can then design a tissue compensator to fit the individual patient.

Humans↗
PubMed1978

A computer-assisted three-dimensional treatment planning system.

The three-dimensional treatment planning system developed at the Rhode Island Hospital visualizes the spatial interrelationships of the radiation beam, the tumor, and the adjacent organs within the patient. It is possible to rotate and vary the scale of the display to better comprehend the extent of these structures. By viewing the display as if from along the radiation beam, one can design shaped treatment fields which best suit the three-dimensional nature of the disease. With this system, it is possible to reduce the volume of normal tissue which would typically be irradiated if two-dimensional treatment planning techniques and assumptions were employed.

Computers↗
PubMed1979

Changing concepts in psychosomatic theory. I. Historical analysis.

Psychosomatic medicine has utilized static, linear, two-dimensional models in attempting to identify a causative relationship between psychosocially conceptualized events and biologically conceptualized ones. This has led to inherent difficulties that are logistically unresolvable. For example, the different language systems lend themselves to overlap with the possibility of addressing themselves to describing manifestations of the same phenomenon from dissimilar conceptual orientations. The problem is further complicated by attempting to link these together in a temporal sequence suggesting a causal relationship. We have ignored both feedback processes in terms of a sequential process modifying a preceding one and the possibility that a reverse order of relationships might explain some processes. In our quest for specificity, we have ignored that disease states are not static ones any more than is life but change over time, suggesting that alterations will occur in the original relationships proposed among our different systems of conceptualization. Among the remedies suggested for a reordering of our thinking are: (a) a revision of linear unidirectional causative models to cyclical bidirectional models; (b) a reformulation of two-dimensional temporal linear relationships toward three-dimensional spherical ones which overlap in time; (c) the relating of these aspects not to each other but to a third factor, common to the three which remains to be identified; (d) the development of a language that bridges the psychological, social and biological.

History, 19th Century↗
PubMed1977

Stereology, a new quantitative morphological approach to study prostatic function and disease.

Although a considerable amount of quantitative biochemical data of the prostatic gland is available the current morphological information is restricted up to now to descriptive findings. Stereology is the application of mathematical axioms and allows one to quantitate three-dimensional structures from the measurement of two-dimensional cross sections thereof. The stereological techniques provide values for volumes, surfaces and number of tissue and glandular cellular components found within the prostatic gland. Four examples are presented to illustrate how quantitative morphology can be applied to study prostatic function and disease. A stereological model, which provides information on the structure of the prostatic gland is shown for the rat prostate (ventral lobe). The model consists of morphologically defined space and membrane components of the prostatic gland and the glandular cell. The alteration, induced in the glandular cells of the ventral lobe of the rat prostate by administration of a synthetic progestine (ethinyl-norgestrienone) were studied by stereological methods. An attempt was made to quantify the morphological aspects of both, the epithelial and stromal components of the normal human prostate (3rd decade of life) and of benign prostatic hyperplasia. In comparing the stereological data of the glandular prostatic cell in the normal human prostate with that in benign prostatic hyperplasia, a diminished secretory activity of the glandular cell in benign prostatic hyperplasia is indicated. A high volume density of the fibromuscular tissue, as well as an activation of the smooth muscle cell in the fibromuscular tissue is observed in benign prostatic hyperplasia.

Adult↗
PubMed1976

A three-dimensional reconstruction of the polygonal pattern on placental coated-vesicle membranes.

There is a surface structure on the coated vesicles of human placenta. Some features of this structure have been examined. Measurements of the polygonal network seen in surface views have been made and compared with measurements of structures projecting from vesicle walls in median sections. A 3-dimensional reconstruction of the vesicle shows the pattern to be one of raised ridges. Use of a goniometer to tilt the specimens has confirmed the assumption that both types of image obtained as from one structure. Although it is usually the case that vesicles are approximately spherical, some are definitely irregularly shaped. For this reason it is suggested that the walls of the polygons need not always be packed into a structure with a regular and precisely maintained pattern. Consideration of the surface structure in the light of current understanding of the cell membrane as a dynamic system leads to a possible explanation of the process of vesicle formation in this context and of the selective nature of uptake by micropinocytosis.

Humans↗
PubMed1979

Three-dimensional reconstruction of the chromatin bodies in the nuclei of mature erythrocytes from the newt Triturus cristatus: the number of nuclear envelope-attachment sites.

The arrangement of the chromatin bodies in the interphase nuclei of 6 erythrocytes has been investigated by means of 3-dimensional reconstruction from electron micrographs of serial sections. When the borders of chromatin bodies are marked on the surface of each model, discrete areas of chromatin in contact with the nuclear envelope are revealed. The number of these areas in approximately equal to the number of chromosomes in the diploid set. The data suggest that each chromatin body corresponds to a condensed interphase chromosome and that each chromosome is attached to one discrete site on the nuclear envelope. The data are insufficient to show whether or not the condensed chromosomes are arranged in any orderly pattern in these nuclei.

Animals↗
PubMed1976

Optimum amino acid complement for protein synthesis by rat mammary cells in tissue culture.

The amino acid requirement of rat mammary cells for milk protein synthesis was investigated in dispersed cell culture. A three-dimensional central composite design utilizing three variables (X1 = lysine; X2= methionine, valine, and arginine; X3 = isoleucine, tryptophan, threonine, phenylalanine, and histidine) at five concentrations each, was duplicated twice with mammary cells from lactating Sprague-Dawley rats. The optimum combination of amino acids for maximum milk protein synthesis from multiple regression models was X1 15.0-, X2 4.5-, and X3 1.5-fold their quantities in Eagle's minimal essential medium with leucine, tyrosine, cystine, and glutamine at the base 1-fold in the medium.

Amino Acids↗
PubMed2025

Proteomics of Duchenne Muscular Dystrophy Patient iPSC-Derived Skeletal Muscle Cells Reveal Differential Expression of Cytoskeletal and Extracellular Matrix Proteins.

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total, 3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9, Collagen 18A, Tropomyosin 1, BASP1, RUVBL1, and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol.

Humans↗
PubMed1978

Assignment of the cysteinyl 13C nuclear magnetic resonances and comparison of other aliphatic amino acid resonances of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins.

13C NMR spectra of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins show that some 13C resonances of the aliphatic amino acid residues are shifted significantly from their corresponding resonance positions in the spectra of model polypeptides or apoferredoxin. Thirteen 13C resonances are shifted into the 80- to 120-ppm (from CS2) region, and have been assigned to the cysteinyl alpha and beta carbon atoms. The remaining shifted resonances in the 120- to 190-ppm region are tentatively assigned to amino acid residues that may be close to [4Fe-4S] clusters of the oxidized and reduced ferredoxins. The similarity in the shift pattern of the corresponding 13C resonances of the cysteinyl alpha and beta carbon atoms in the three ferredoxins studied suggests that the three-dimensional amino acid environments of the corresponding [4Fe-4S] clusters in each protein are similar.

Amino Acids↗
PubMed1977

Biomechanical analysis of static forces in the thumb during hand function.

Using a three-dimensional analysis, the internal forces in the joints and soft tissues of the thumb during pinch and grasp were calculated. To do this, mechanical equivalents were ascribed to the anatomical system, the joint orientation and tendon locations were determined from biplanar roentgenograms of five marked normal cadaver specimens, and the magnitudes of forces in the tendons, intrinsic muscles, joint contact surfaces, and constraining ligaments were calculated based on assumed loads applied to the tip of the thumb in various types of pinch and grasp. These results are the direct extension of a two-dimensional analysis that proved inadequate for the determination of static tendon and joint forces. Extrinsic and intrinsic tendons of the thumb were found to sustain forces of up to 10.0 and thirty kilograms during pinch, producing five kilograms of force at the thumb tip and forces of as much as fifty kilograms during grasp. The joint compression (contact) forces averaged three kilograms of force at the interphalangeal joint, 5.4 kilograms at the metacarpophalangeal joint, and 12.0 kilograms at the carpometacarpal joint during simple pinch (one kilogram of applied force). Compression forces of as much as 120 kilograms may occur at the carpometacarpal joint during strong grasp.

Biomechanical Phenomena↗