Note on the analysis of thymidine labeling in cells with chromosome-number dispersion

A method of analyzing thymidine labeling in a population of cells is formulated. The formulation establishes a unique relation between a specific set of labeling data and a specific set of cells in the population, viz. that set of cells having a particular chromosome number. The analysis employs a cell-state variable, i.e., a quantity which specifies the progress of a cell through its lifecycle. This variable is defined in terms of the nucleo-protein content and configuration of the chromosomes. The relation mentioned above leads immediately to an expression for the number of cells present at a particular time following labeling which have a given amount of label per cell and a given chromosome number.     

A new method for morphometric analysis of tissue distribution of mobile cells in relation to immobile tissue structures

The distribution of cells in stained tissue sections provides information that may be analyzed by means of morphometric computation. We developed an algorithm for automated analysis for the purpose of answering questions pertaining to the relative densities of wandering cells in the vicinity of comparatively immobile tissue structures such as vessels or tumors. As an example, we present the analysis of distribution of CD56-positive cells and of CXCR3-positive cells (relative densities of peri-vascular versus non-vascular cell populations) in relation to the endothelium of capillaries and venules of human parietal decidua tissue of first trimester pregnancy. In addition, the distribution of CD56-positive cells (mostly uterine NK cells) in relation to spiral arteries is analyzed. The image analysis is based on microphotographs of two-color immunohistological stainings. Discrete distances (10-50 μm) from the fixed structures were chosen for the purpose of defining the extent of neighborhood areas. For the sake of better comparison of cell distributions at different overall cell densities a model of random distribution of "cells" in relation to neighborhood areas and rest decidua of a specific sample was built. In the chosen instances, we found increased perivascular density of CD56-positive cells and of CXCR3-positive cells. In contrast, no accumulation of CD56-positive cells was found in the neighborhood of spiral arteries.     

Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

Summary— An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

Allometric relations of total volumes of prolactin cells and corticotropic cells to body length in the annual cyprinodont Cynolebias whitei: effects of environmental salinity,stress and ageing

Summary An analysis of the allometric relations of the total volumes occupied by prolactin (PRL) and corticotropic (ACTH) cells (PRL volume and ACTH volume, respectively) to body length and a study of the immunocytochemical staining intensity of PRL and ACTH cells were used to determine the differences in activity of PRL and ACTH cells in freshwater-reared and in saltwater-reared Cynolebias whitei during the entire lifespan of this annual cyprinodont fish. An inflection in the allometric relation of PRL volume to body length was observed in fish of one-week old. The relatively large PRL volume in younger fish may be related to PRL cell activity before hatching. No inflections were observed in the allometric relations of PRL volume and ACTH volume to body length at the onset of maturation and the onset of ageing, indicating that the increased pituitary growth in maturing and ageing C. whitei is not the result of changes in PRL or ACTH cells. The slope of the allometric relation of PRL volume to body length in freshwater-reared fish was significantly steeper than the slope in saltwater-reared fish. The PRL volume in adult freshwater-reared fish was eight times larger than that in saltwater-reared fish of the same length. The intensity of immunocytochemical staining of saltwater PRL cells was significantly reduced. These volumetric and staining differences correspond to the low functional demand put upon PRL cells in saltwater-adapted fish. In contrast, the slope of the allometric relation of ACTH volume to body length and the intensity of immunocytochemical staining of ACTH cells were similar in freshwater-reared and in saltwater-reared fish. It is concluded that the functional demand put upon ACTH cells is similar in freshwater-reared and saltwater-reared C. whitei; the involvement of ACTH cells in the osmoregulation of the fish in both environments is similar.     

PC-1、EphA3和SGEF蛋白表达的相互影响

目的:在293T和LNCaP细胞中验证PC-1、EphA3和SGEF蛋白表达的相互影响。方法 :采用半定量RT-PCR及Western印迹检测EphA3和SGEF在LNCaP细胞中与PC-1的关系,分别在LNCaP和293T细胞中检测EphA3对SGEF表达的影响。结果:在LNCaP细胞中,EphA3和SGEF在RNA及蛋白水平上均受PC-1高表达诱导,而EphA3的高表达对SGEF的表达无明显影响;但在293T细胞中,SGEF受EphA3表达水平的影响,随EphA3表达量的增加而升高。结论:PC-1、EphA3与SGEF蛋白表达的相互影响与细胞类型相关。     

Noise generation, amplification and propagation in chemotactic signaling systems of living cells

Theoretical considerations of stochastic signal transduction in living cells have revealed the gain-fluctuation relation, which provides a theoretical framework to describe quantitatively how noise is generated, amplified and propagated along a signaling cascade in living cells. We chose the chemotactic signaling of bacteria and eukaryotic cells as a typical example of noisy signal transduction and applied the gain-fluctuation relation to these signaling systems in order to analyze the effects of noise on signal transduction. Comparing our theoretical analysis with the experimental results of chemotaxis in bacteria Escherichia coli and eukaryote Dictyostelium discoideum revealed that noise in signal transduction systems limits the cells' chemotactic ability and contributes to their behavioral variability. Based on the kinetic properties of signaling molecules in living cells, the gain-fluctuation relation can quantitatively explain stochastic cellular behaviors.     

The migration of dermal cells during blastema formation in axolotls

Using the diploid/triploid cell marker in the axolotl (Ambystoma mexicanum) we have examined the movement of cells from the dermis into the early limb blastema. Cells of dermal origin begin to migrate beneath the wound epithelium at about 5 days postamputation, and by 10 days they are widely distributed across the amputation surface. By 15 days, a dense accumulation of blastema cells is present beneath the apical cap, and these cells are preferentially oriented in a circumferential direction. These results are discussed in relation to previous studies showing that the progeny of dermal cells become widely distributed during regeneration, and that cells of dermal origin are a major source of blastema cells. The results are also discussed in relation to ideas about how growth and patterning of the new appendage occur.     

Changes in the intracellular concentrations of adenosine phosphates and nicotinamide nucleotides during the aerobic growth cycle of yeast on different carbon sources

1. Methods for the quantitative extraction of adenosine phosphates and nicotinamide nucleotides from yeast cells are described. 2. The intracellular concentrations of adenosine phosphates and nicotinamide nucleotides were measured during the aerobic growth cycle of yeast on glucose and galactose. 3. When sugars were still present in the media the intracellular concentrations of NADH and AMP were in general higher in glucose- than in galactose-grown cells, whereas ADP concentration was always lower in glucose-grown cells. 4. The adenylate-kinase reaction was found to be far from equilibrium in the glucose-grown cells and when glucose was still present in the growth medium. 5. The significance of the changes in the intracellular concentrations of adenosine phosphates and nicotinamide nucleotides observed during growth on either sugar is discussed in relation to the metabolism and growth of the cells. 6. The differences observed in the concentrations of these cofactors in glucose- and galactose-grown cells are also discussed in relation to the type of metabolism of these cells. Control of glycolysis at the level of phosphofructokinase in galactose-grown cells and at the level of phosphoglycerate kinase in glucose-grown cells is suggested. 7. ADP is suggested to be the inducer of formation of respiratory enzymes.     

Ultrastructure of the branchial epithelium of an amphibious brackish-water crab

The ultrastructure of the branchial epithelium of the amphibious brackish-water crab Uca mordax (Smith) was investigated in relation to adaptation to the salinity of the medium. No distinct differences were observed in the epithelial structure of animals adapted to either 100% sea water or to 1% sea water. Thus any interpretation of the significance of particular structures in relation to specific transport processes should be regarded with caution. Apart from strict epithelial cells, pillar cells and glycogen (presumed) storage cells were found. The epithelial cells showed very well-developed apical microvilli or lamellae and basal interdigitations with adjacent cells. Well-developed junctional complexes were seen (band desmosomes, septate desmosomes, gap junctions). The cells are extremely rich in mitochondria. Microtubules, peroxisome-like bodies, multivesicular bodies and near-nuclear Golgi complexes were present.     

Mutually exclusive expression of fibronectin and glial fibrillary acidic protein in cultured brain cells

The reported expression of the cell surface-associated, mainly mesenchymal glycoprotein fibronectin by cultured glial cells is in discrepancy with recent work on brain tissue failing to demonstrate any glial or neuronal fibronectin. We have investigated the expression of fibronectin in relation to glial fibrillary acidic protein in cultured human glial and glioma cell lines as well as in cultures derived from newborn rat brain. Using double immunofluorescence technique we found that cells containing glial fibrillary acidic protein do not express fibronectin, and vice versa. The only exception to this rule was the occasional finding of fibronectin at points of cell-to-cell adhesion also in relation to cells containing glial fibrillary acidic protein. The results were also tested by polyacrylamide gel electrophoresis of the culture media of the human cell lines, and by subcultures from the brain of newborn rat, cultures stimulated with dibutyryl cyclic AMP (db-cAMP), and by vinblastine treatment of the cells. The lack of expression of fibronectin in cells containing glial fibrillary acidic protein, a gliospecific cytoskeletal protein, is discussed with reference to glio-mesenchymal interactions and glial markers in vitro.     

Conception of contact potential differences in the bioelectrochemistry phenomena concerning cellsin Vitro

Summary The contact potential difference is presented in relation to the electrokinetic potential and the surface potentials of the cells.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

The simultaneous exposition of galactose and mannose-specific receptors on rat liver macrophages is developmentally regulated

We studied the simultaneous binding of galactose and mannose-exposing ligands in sinusoidal rat liver cells during development and aging. The galactose-specific receptors were visualized using 17 nm diameter colloidal gold particles coupled with Lactosylated bovine serum albumine (LacBSA), while mannose-specific receptors were localized by means of 5 nm diameter particles adsorbed with mannan. We observed the presence of four different classes of Kupffer cells in relation to the ligands bound. The percentage of each group of Kupffer cells varied in relation to the age of the subject from which the sample was taken. There were few double-labelled cells in the livers from newborn rats, with numbers increasing with age to adulthood, and decreasing again in the older animals. Cells without labelling were in the majority after birth, but they decreased in number up to adulthood and increased again during subsequent aging. The numbers of single-labelled cells did not change significantly during liver maturation. We hypothesize that the exposition of galactose and mannose-specific receptorial systems is regulated by developmental conditions.     

Fibronectin in the area opaca of the young chick embryo

Summary Distribution of fibronectin-like immunoreactivity was studied in the area opaca of the young chick embryo (stages 4–6 HH) by use of the immunofluorescence and protein A-coupled to colloidal gold techniques. Fibronectin, associated to the basement membrane, formed a fibrillar network, the pattern of which changed from the centre to the periphery of the area opaca. At the ultrastructural level, differences in fibronectin distribution were found between non-moving and moving cells. The epithelial-like cells presented fibronectin staining exclusively on their basal side. Actively migrating cells (edge and mesodermal cells) showed immunoreactive material localized around their entire surface and within the cytoplasm. The fibronectin distribution is discussed in relation to three important phenomena taking place during the early growth of the area opaca: (i) anchorage and migration of the edge cells, (ii) modification of cell shape in relation to mechanical tension, and (iii) expansion of the area vasculosa.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The pars intermedia of Sarotherodon mossambicus contains two structurally different endocrine cell types. The predominant cell type is assumed to synthesize MSH and related peptides. The second cell type is PAS positive; its function and products are unknown. In this second cell type changes occur in relation to background colour and illumination. Thus, PAS positive cells of fish adapted to a white background are less numerous and metabolically less active than those of fish adapted to a black background, and are most active in fish kept in total darkness. In blinded fish, whether in light or in darkness, the activity of the PAS positive cells is similar to that of the black background-adapted animals. The significance of these responses in relation to the control of background adaptation is discussed.     

Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.     

Does adipocyte hypercellularity in obesity exist?

Adipose tissue samples were biopsied from three subcutaneous sites in 80 obese and 27 non-obese patients. Additional samples were taken from intra-abdominal sites in 44 of the patients. There was a small increase in the calculated number of fat cells in the more obese patients, but there was no relation between fat-cell number and obesity of childhood onset. Omental fat cells were one-third the size of subcutaneous cells. Thus the calculated number of fat cells, usually based solely on subcutaneous samples, is an underestimate of the true number, and most obese patients can accommodate their fat without needing to recruit new cells. The diagnosis of "hyperplastic" obesity--that is, an excess number of fat cells--is unreliable and its relation to infantile obesity doubtful.     

Splenomegaly reaction of the chick embryo following chorio-allantoid graft of chicken-spleen fragments]

The spleen enhancement reaction of the chick embryo, following the insertion (grafting or injection procedure) of homologous spleen cells is one of the results of the graft-versus-host reaction (G.V.H. reaction). Irrespective of the usual kinds of G.V.H. reaction measured, it has been proved that the relation between the number of immuno competent cells and the reaction intensity is linear. Our study shows that the relation is not the same when the chorio-allantois membrane grafting procedure is used instead of injection into the veins. However, two facts remain unchanged 1) the minimal amount of spleen cells sufficient to provoke a spleen enhancement is low, 2) there is a link between the number of homologous spleen cells and the rate of spleen enhancement, but in this case it was not shown to be linear. In the light of this, the role played by the chorio-allantois membrane is being debated.     

Activation of c-MET induces a stem-like phenotype in human prostate cancer

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway.Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 ('stem-like signature'). We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f.In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.