The relationship of cellular structure and function: the matrix system.
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The growth-enhancing effect and the differentiation of colonies formed by native and frozen-thawed myelokaryocytes in semiliquid agar were observed over a period from the 2nd to the 28th day. The cultivation of bone marrow in agar was shown to form colonies consisting of haemopoietic cells as well as of stromal elements. Under the cultivation of frozen-thawed bone marrow, the retardation of proliferative activity of growing cells was observed within up to 4 days. Later on the proliferative character and the rate of the myelokaryocytes of both the types were seen identical. The question of a possible existence of haemopoietic tissue and stromal elements in the progenitor cells of normal bone marrow is being discussed.
Investigation of the dog's insular claustrum was performed using morphometric methods. The brains of 14 dogs (7-14 year-old) were studied. The size, shape and density of neurons are different in various parts of the insular claustrum. The posterior part, related to the visual system, is heterogenous and possesses medium-sized and small densely packed neurons, the average cross-sectional area of cell body being 175.8 +/- 45.5 micron2. The similar average cross-sectional area of cell body (174.1 +/- 55.3 micron2) was found in the central part of the insular claustrum. However, neuronal architecture is here less heterogenous and neurons are less densely arranged. The anterior part, connected mainly with the frontal and motor cortex, is more homogenous and contains loosely arranged neurons of a rather large size (average size--198.7 +/- 53.46 micron2).
Five of six Baculovirus species studied induce cytoplasmic and nuclear fibrous sheets, observed by electron microscopy, within infected cells of lepidopterous. The reticulated structures, revealed by fluorescence microscopy, assimilate the fibrillar substance induced by these viruses thanks to the affinity they show for globulins of healthy rabbits.
Fatty acid composition of lipopolysaccharides (LPS) and cytoplasmic membranes (CPM) of Yersinia pestis [correction of the plaque microbe] has been studied. Concentration of certain acids in the LPS content proved sharply different and thus the strains were separated into groups. High content of 3-oxymiristinic acid was a distinction of vaccine strains. Virulent strains of one of the groups regularly differed from the rest of virulent strains by high content of laurinic acid. Laurinic and two nonidentified acids, which were not found in the virulent cultures, were present in CMP lipids of vaccine strains.
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Dynamics of substrate pools and level of inorganic phosphate P(i) in the course of glucose-6-phosphate (G6P) utilization by Escherichia coli have been studied by 31P NMR and EPR methods on cells immobilised in a three component water-in-oil microemulsion composed from tridecane, nonionic surfactant Tween 81 and a low amount of water. The state of the electron transport chain in cell membranes has been evaluated on the basis of reduction rates of exogenous nitroxide radicals. Low water activity a(w) in the external microemulsion appears to inhibit, to some extent, glycolytic enzymes and electron transport proteins in the membrane structures without disturbing the membrane integrity and solubilization of lipoproteins in reverse micelles. Enzyme activity has been restricted by the micellar substrate diffusion and by decrease in water activity a(w) in the course of the extensive molecular interchange of bound water between the external microemulsion phase and the internal cell structures.
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New data on the functional properties of the cell structures of cultured T. pallidum were obtained, which permitted the construction of new more effective preparations for the diagnostics of syphilis. T. pallidum cell walls were used as antigen in the complement fixation test (CFT) and the development of a new enzyme immunoassay system for the determination of IgM and IgG antibodies to T. pallidum with the visual evaluation of results, while T. pallidum cytoplasm was used as sorbent in the immunofluorescence test (IFT-abs) for the serodiagnosis of syphilis. The immunization of rabbits with T. pallidum cell walls increased the titers of positive control sera used in CFT for diagnosing syphilis.
Complex study of the structure of islands of Calleja in carnivore brain (cat and dog) was conducted. Using Nissl and Golgi methods, HRP axonal transport and electron microscopy the islands were found to be composed of cells of 3 types varying in size, shape, dendrite spatial distribution and ultrastructure. The majority of cell population in the islands is formed by small granular cells with scarce medium-sized, mitral-like and larger cells among them. The administration of retrograde marker into posterolateral hypothalamus provided the evidence for the presence of cells in the islands of Calleja projecting to this area. These were found to be mitral-like cells. Other cells did not exhibit staining. The resemblance between mitral-like and granular cells of the large islands of Calleja with mitral-like and granular cells of olfactory bulb, respectively, supports our assumption that these islands compose the central sensory nucleus (presumably of the sensory terminal nerve).
After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or--by sophisticated pattern recognition methods--identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are presented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data.
Diffusion-weighted imaging (DWI) provides a unique form of magnetic resonance (MR) contrast that enables the diffusional motion of water molecules to be quantitatively measured. As a consequence, DWI provides information about the orientation, size and geometry of brain structures. Cellular structures in the central nervous system restrict water molecular motion, and the apparent diffusion coefficient (ADC) is reduced compared to diffusion in bulk water. Pathological processes that modify tissue integrity, thus removing some of the "restricting" barriers, can result in increased ADC. Preliminary studies in multiple sclerosis (MS) using DWI showed that the ADC is higher in macroscopic lesions than in the normal appearing white matter (NAWM). The ADC is also dependent on the direction in which diffusion is measured, thus making comparison of ADC values meaningless without taking into account the measurement direction. One measurement of diffusion that is independent of the orientation of structures is provided by measuring the ADC in three orthogonal directions, and then averaging the results to form the mean diffusivity, D. We obtained DW scans from 35 patients with relapsing-remitting MS and 24 healthy volunteers. D was measured inside T2-visible lesions and regions located in different areas of the NAWM. D histograms from a large portion of the brain were created. MS lesions had a significantly higher D than NAWM. T1-hypointense lesions had the highest diffusion values, consistent with more severe tissue disruption. D was higher in the NAWM from patients than in the white matter from healthy controls. We also found significant differences between D histogram-derived measures from patients and controls, confirming the presence of diffuse damage in the brain of patients with MS.
Although brain studies began in ancient Egypt, speculations on vertebrate brain evolution occurred only much later, after the publication of Darwin's Origin of Species in 1859. Subsequently, views of brain evolution have been shaped by a complex interplay of theory and technique. Darwin's theory allowed the variation in brain size and complexity to be re-interpreted within an evolutionary context, albeit an erroneous pre-Darwinian context based on scala naturae. With the development of histological techniques, research shifted to descriptions of cellular structure, cellular aggregates and their putative interconnections. In spite of these technical advances, brain evolution continued to be viewed within the context of scala naturae. Following the publication of The Comparative Anatomy of the Nervous System of Vertebrates by Ariëns Kappers, Huber, and Crosby in 1936, there followed a period of stasis, after which biological views of evolution were radically altered by the confluence of genetics, paleontology, and systematics, termed the Evolutionary Synthesis. Against this background, the development of new experimental techniques for establishing neural connections resulted in a new flowering of comparative neuroanatomy. While comparative descriptive and experimental studies of brain organization continue, the rapprochement of embryology and genetics is fueling a new renaissance that promises to increase our understanding of brain evolution and its genetic basis.
Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.
Cystinuria was first described in 1810. Since the initial description, a group of renal cellular transport deficit diseases has been characterized. The study of genetic diseases that create a specific aminoaciduria has expanded our knowledge of cellular structure, cellular transport, and intracellular concentration gradients. A review of the theories and experimental data obtained through investigations of the renal aminoacidurias are presented.
Biological cells are complex in both morphological and biochemical structure. The effects of cellular fine structure on light scattered from cells are studied by employing a three-dimensional code named AETHER which solves the full set of Maxwell equations by using the finite-difference time-domain method. It is shown that changes in cellular fine structure can cause significant changes in the scattered light pattern over particular scattering angles. These changes potentially provide the possibility for distinguishability of cellular intrastructures. The effects that features of different intrastructure have on scattered light are discussed from the viewpoint of diagnosing cellular fine structure. Finally, we discuss scattered light patterns for lymphocyte-like cells and basophil-like cells.
Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.
The cellular target structures for six monoclonal antibodies raised against cultured human bladder carcinoma cells (TCC) were investigated. The specificities of these antibodies when tested against a large panel of cells have been described in the companion paper. Radiolabeled cell lysates were precipitated with the different monoclonal antibodies bound to protein A (Staphylococcus aureus) on a matrix of Sepharose beads. The precipitates were separated by sodium dodecyl sulfate- gel electrophoresis (SDS-PAGE) and analyzed by autoradiography. The antibodies 4B5, 7E9, and 14B11 have previously been found to react in a similar way with TCC-targets and some non-TCC tumor cells, but not with normal urothelial cells or cells of hematopoietic origin. When tested with lysates of a TCC-cell line (TCCSuP) a strong 92K band and a weak 23K band were precipitated with any one of these antibodies. These polypeptides were expressed on the cell surface and were not linked by disulfide bonds. Depletion experiments confirmed that the three antibodies recognized the same antigens. Another antibody (4E8) probably directed to a differentiation antigen present on both urothelial and melanoma cells detected two high molecular polypeptides, 190K and 170K. Antibodies from the S2C6 hybridoma, which displayed a distinct dual specificity for TCC- targets and for malignant or transformed cells of B cell origin, precipitated a 50K component from extracts of either TCC- or B cell-derived cell lines. Antibodies produced by the S2A9 hybridoma were shown to bind to a framework epitope of the HLA-A, B, C heavy chain.