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RNA X DNA in situ hybridization revealed a high level of ETn ("early transposon") transcripts in the pluripotent cell lineage of the 3.5- to 7.5-day mouse embryo. Some extra-embryonic ectoderm derivatives also show a high level of ETn transcripts at these stages. Older embryos (8.5 days and later) have a uniform low level of ETn transcripts.
Ca2+ imaging and single-channel recording were used to study the regulation of cytosolic free Ca2+ ([Ca2+]i) in local regions of frog sympathetic neurons. Digital imaging with the fluorescent Ca2+ indicator fura-2 demonstrated: (i) resting [Ca2+]i of 70-100 nM; (ii) significant increases in [Ca2+]i in growth cones and cell bodies following depolarization induced by extracellular electrical stimulation or increased external K+; (iii) in cell bodies, large transient increases in [Ca2+]i following exposure to caffeine and sustained oscillations in [Ca2+]i in the presence of elevated K+ and caffeine; and (iv) in growth cones, smaller and briefer changes in [Ca2+]i in response to caffeine. The nature of the depolarization-induced Ca2+ entry was studied with cell-attached patch recordings (110 mM Ba2+ in recording pipette). Ca2+ channel activity was observed in 18 of 20 patches on cell bodies, 3 of 5 patches along neurites, and 36 of 41 patch recordings from growth cones. We observed two types of Ca2+ channels: L-type channels, characterized by a 28-pS slope conductance, sensitivity to dihydropyridine Ca2+ channel agonist, and availability even with depolarizing holding potentials; and N-type channels, characterized by a 15-pS slope conductance, resistance to dihydropyridines, and inactivation with depolarized holding potentials. Both types of channels were found on growth cones and along neurites as well as on cell bodies; channels often appeared concentrated in local hot spots, sometimes dominated by one channel type.
The multiplication in gases of ionization electrons, by the effect of the electric fields between parallel electrodes, leads to the emission of light from the molecules excited in the avalanche process. The optical imaging of this light, with intensifiers, on charge-coupled devices permits the localization, in the gaseous volume, of the entrance points of the beta-particles emitted by radioactive compounds placed close to or at the cathode electrode. Thin slices of anatomical samples labeled with 3H show detailed structures 30 microns in size. Gels carrying 32P or 35S are imaged with accuracies of the order of 0.5 mm (full width at half maximum). In comparison with photographic emulsion, the gain in time for data taking is close to a factor of 100, with the advantage of linearity and wider dynamic range in the intensity measurement and a greatly improved signal-to-noise ratio.
The malignant CHO-K1 cell is reverse-transformed by cAMP, regaining the phenotype of a normal fibroblast. During this reaction, much of its DNA re-acquires sensitivity to hydrolysis by DNase I in a way characteristic of the normal fibroblast. Exposed DNA forms a rim about the nucleus in both the normal and reverse-transformed cell but not in the malignant CHO-K1. Reacquisition of the nuclear rim requires an organized cytoskeleton. Sequestered DNA forms families of different degrees of sequestration. In accordance with previous theoretical developments it is proposed that (i) genes specific to a given differentiation state are stored in the nuclear rim, whereas genes specific to other states are sequestered within the nucleus; (ii) only exposed genes are active, and their activity is modulated by regulatory molecules in the fluid medium; (iii) exposure and sequestration are regulated by cytoskeletal and nuclear protein structures; (iv) in at least several types of cancer the regulatory defect lies in the genome exposure process so that the specific DNA sequences and their associated growth regulatory loci have been transferred from the exposed to the sequestered condition with consequent loss of the nuclear rim of exposed DNA. The methodology described should be generally applicable to examining the accessibility state of subsets of DNA during various physiological modulations of cell function.
We demonstrated that two mRNAs that are synthesized during the vitellogenic period of oogenesis and that code for cell surface proteins are asymmetrically distributed in the unfertilized egg of Paracentrotus lividus. At fertilization, these RNAs rapidly localize in the cortical zone at the animal pole of the egg. They are then detected in the mesomeres and the macromeres, but not in the micromeres, and thereafter are found in the ectoderm but not in the vegetal plate, mesenchyme cells, or early intestine. They disappear in late gastrula. The proteins synthesized by these mRNAs show the same territorial location during the period examined here, which included the unfertilized egg and the 16-blastomere stage. These conclusions were reached on the basis of in situ hybridization and immunostaining experiments, as well as Northern and Western blot analyses of isolated blastomeres. The possible significance of this asymmetric distribution of these two mRNAs and proteins in the establishment of the animal/vegetal axis is discussed.
PURPOSE: To estimate the distributions in width as well as in density of macular pigment (MP) in humans at different ages, imaging fundus reflectometry was employed as a tool to reach our aim. METHODS: Imaging fundus reflectometry was used to measure the macular pigment distribution in human eyes with a modified fundus camera to which a cooled CCD camera was attached. The fundus images were taken at 460 nm and 560 nm after the retina was light-adapted to completely bleach rhodopsin. The density of macular pigment was estimated using a model described in the text. The variation in density with retinal eccentricity was fit to Gaussian distribution. The width of MP in retinal eccentricity was estimated by 95% covering area of the distribution profile. A total of 54 normal people served as subjects. They were divided into three groups with ages of 24.8 +/- 2.6 years (N = 24), 40.2 +/- 8.3 years (N = 13) and 67.5 +/- 7.1 years (N = 17) respectively. Statistical t-tests were employed to evaluate the differences in MP density, the half width of MP distributed (HWMPD) in retina with respect to age between the groups. Linear regression was also applied to reveal the relationships of HWMPD distribution with respect to age. RESULTS: The results indicate that the average MP densities are 0.23 +/- 0.08, 0.22 +/- 0.06 and 0.23 +/- 0.06 density unit (DU) for the young to old age groups. The average MP density is 0.23 +/- 0.07. No statistical significant difference is found in the peak MP density between the groups (all p > 0.05). This result pretty agrees with the data that obtained from others by using optical methods. For the young to old age groups, the average estimated HWMPD are 2.6 degrees +/- 0.5 degrees, 3.1 degrees +/- 0.5 degrees and 4.1 degrees +/- 0.6 degrees respectively. The differences between loci of HWMPD in the three groups are statistically significant with p = 0.0059, p < 0.0001 and p = 0.0001 for young vs. mid-age, young vs. old and mid-age vs. old respectively. Linear regressions for the HWMPD vs. age (r = 0.734, p < 0.001) implies that the macular pigment can gradually accumulate and spread out to the periphery of retina through our life span with an increase rate of approximately 0.03 degrees /year. CONCLUSIONS: The accumulation of MP is reflected by the broader area in the macula lutea due to age, but no significant change is observed in peak density. The cause of the extension in the macular area with respect to age is unknown.
The glands in the human esophageal mucosa have been considered rudimentary, and have been poorly studied. Only recently, their role in the defense of the esophageal mucosa in gastroesophageal reflux disease has been put in evidence. In the present study, the presence of the different esophageal gland types was observed in 82 necropsy specimens. A precise topographical study was possible in 15 specimens. Cell proliferation parameters, namely DNA synthesis and mitotic index values in the squamous epithelium and in the esophageal glands were measured in 8 patients undergoing a surgical palliative esophagectomy, after in vivo labelling with bromodeoxyuridine. Upper mucosal, lower mucosal, and submucosal glands were observed in respectively 3.7%, 87% and 99% of the 82 esophageal specimens. Our topographical analysis showed that the upper mucosal glands, lower mucosal glands, and submucosal glands are occupying respectively 0.02%, 2.61%, and 3.96% of the esophageal surface. DNA synthesis and mitotic index values in the progenitor zone of the squamous epithelium were respectively 7.91% and 0.81%, whereas the proliferative activity in the glands was extremely low. The labelling index In the excretory ducts of the glands was only 0.07%, while no labelled cells nor mitotic figures were observed in any of the glandular acini.
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BACKGROUND: Although apoE-/- mice are characterized by hypercholesterolemia, the bile acid enterohepatic circulation, which plays a crucial role in cholesterol homeostasis, has not been examined in these mice. The differences between apoE-/- and C57BL/6 mice in expression of the ileal ASBT and ILBP and in intestinal bile acid absorption were studied. METHODS: The intestinal tissues of the fetal, neonatal and post-weaning mice were processed for immunohistochemistry. Body retention and fecal excretion of 75SeHCAT were measured. The bile acid pool size and its composition were analysed by HPLC. RESULTS: In apoE-/- and C57BL/6 mice, the bile acid pool size was 75 +/- 13 and 78 +/- 13 micromol/ 100 g body weight, respectively, while the ratio of cholic acid/beta-muricholic acid was 1.8 +/- 0.3 and 1.4 +/- 0.3 (P < 0.05), respectively. The daily body retention of 75SeHCAT was 48% = 1.8% in C57 black mice and 58.4% +/- 2.7% in apoE-/- mice (P < 0.05). In both mouse strains, ASBT expression in the small intestine was found in the near-term fetal and post-weaning mice, while ILBP expression was found in all postnatal mice. In the post-weaning mice, ILBP expression was limited to the distal 25%-30% of the small intestine, while ASBT expression was limited to the distal 18%. CONCLUSIONS: The bile acid enterohepatic circulation in apoE-/- mice probably does not differ greatly from that in C57BL/6 mice.
Tuberculosis caused by Mycobacterium bovis was diagnosed in 36 of 68 (53%) brushtail possums (Trichosurus vulpecula) trapped in August 1992 from a population of exceptionally low density (trap catch <3%) on a forest-scrub margin in Westland, New Zealand. The prevalence of tuberculosis in possums, based solely on gross lesions, was at least twice that previously recorded in New Zealand, and was about seven times that recorded from the same population in 1980. More male (66%) than female (33%) possums had grossly visible tuberculous lesions. The distribution of infection appeared continuous along the forest-scrub margin. Both stoats (Mustela erminea) and one of six hares (Lepus europaeus occidentalis) trapped were also infected with M. bovis.
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Poliomyelitis (polio) still remains a great public health problem in developing countries. A study of the distribution of 2205 cases of paralytic poliomyelitis seen in Ibadan City, Nigeria over a 15-year period shows that the incidence of polio is much higher in the central core area of the city where sanitary conditions are poor, whereas in the peripheral parts of the city, where environmental sanitation is better, the incidence of polio is lower. A total of 88.4% of the cases originated from the central core area whereas only 11.6% were from the peripheral area. The polio victims from the peripheral area were, however, disproportionately older than the victims from the core area. It is suggested that in the central core area of Ibadan, where children with the highest risk of polio paralysis reside, immunization against polio should be started much earlier, preferably at birth, while children in the peripheral area who may receive their immunization at a later age (6 months and above) will require booster doses at age 15-18 months and at 5 years of age.
Vase-shaped standard apple trees cv. McIntosh were sprayed with azinphos-methyl at pink, pink and 1st cover and 1st cover only. Residue analyses by gas chromatography revealed detectable residues on foliage until mid summer. At harvest, negligible residue levels were found on the peel and the whole apple. On four trees, fluorescein was sprayed in the same manner as the insecticide and maximum levels of the dye were detected on the outside lower canopy along the row. Minimal concentration of fluorescein was detected on the inner upper canopy away from the direction of the row.
Samples of human rib were collected at autopsy and 20 were selected for marrow fat cell measurement, representing an age range of 16-96 years. The mean diameter of fat cells in red bone marrow of human rib was found to increase from around 48 microns at ages 16-29 years to around 65 microns at ages 82-96 years. There was a greater number of fat cells of smaller size range in younger ages compared with that in older ones. The maximum size of fat cells was found to be 102 microns. Calculated radon-derived doses to haemopoietic tissue ranged from 60 to 162 microSv y-1 at average UK exposures of 20 Bq m-3. It was concluded that the bone marrow fat fraction is the important parameter as far as alpha-radiation dose from radon in fat is concerned. This updates the theoretical estimates of dose carried out by Richardson et al. (1991).
The main intrinsic polypeptide (MIP) is the major protein present in the lens fiber cell membrane and is the product of a gene which, as far as is known, is expressed only in the lens. We have used in situ hybridization and immunofluorescence microscopy to characterize the expression of this gene during the course of development in the rat. At progressive stages of lens morphogenesis, we find that synthesis of the protein is closely tied to the accumulation of MIP mRNA in cells that are committed to terminal differentiation, first in the elongating presumptive primary lens fibers and later in the secondary fibers as they differentiate from the anterior epithelial cells. The transcripts accumulate in the basal cytoplasm of the primary fibers and in the cytoplasm which surrounds the cell nucleus in the secondary fibers. We have compared this pattern of expression with that of a gene for a cytoplasmic protein, beta-crystallin beta-A1/A3. In sharp contrast to the localized concentrations seen for the MIP mRNA, beta-A1/A3 transcripts are relatively uniformly distributed throughout the cytoplasm. Neither MIP nor crystallin gene appears to be transcriptionally active in the undifferentiated epithelial cell, but transcripts from the beta-A1/A3 gene appear earlier in fiber cell differentiation than do those from the gene for MIP.
Chemoattractants stimulate actin polymerization in lamellipodia of polymorphonuclear leukocytes. We find that removal of chemoattractant results in rapid (within 10 s at 37 degrees C) and selective depolymerization of the F-actin located in lamellipodia. Addition of 10 microM cytochalasin B, in the presence of chemoattractant, also resulted in rapid and selective depolymerization of lamellar F-actin. The elevated F-actin level induced by chemoattractant rapidly returns to the level present in unstimulated cells after (a) a 10-fold decrease in chemoattractant concentration; (b) the addition of 10 microM cytochalasin B; or (c) cooling to 4 degrees C. The F-actin levels of unstimulated cells are only slightly affected by these treatments. Based on the similar effects of cytochalasin addition and chemoattractant dilution, it is likely that both treatments result in actin depolymerization from the pointed ends of filaments. Based on our results we propose that chemoattractant-stimulated polymorphonuclear leukocytes contain two distinct populations of actin filaments. The actin filaments within the lamellipodia are highly labile and in the continued presence of chemoattractant these filaments are rapidly turning over, continually polymerizing at their plus (barbed) ends, and depolymerizing at their minus ends. In contrast, the cortical F-actin filaments of both stimulated and unstimulated cells are differentially stable.
p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.
Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin beta by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.