Cell kinetics of vocal fold epithelium in rats.

In order to investigate the kinetics of vocal fold epithelium a bromodeoxyuridine-anti bromodeoxyuridine method has been applied in vivo at both light and electron microscopy level. This method is able to define the length of both epithelium turnover and cell-cycle in basal elements, as well as the existence of a higher proliferation rate during night time in comparison with day time. Moreover distinct labeling patterns observed in incorporating cells allow us to define the precise localization in S-phase of cycling elements.     

A study of photoreceptor-retinal pigment epithelium complex: age-related changes in monkeys

Retinas of 4-, 10-, and 20-year-old monkeys were studied by light microscopy, electron microscopy, and scanning electron microscopy. Sections from the midperipheral region of every retina were selected for comparison. Although no significant differences were found between 4- and 10-year-old retinas, four major changes were found in 20-year-old monkey retinas: (i) increased number of displaced photoreceptor cells (DPC), (ii) increased number of macrophages of different morphology in subretinal space, (iii) increase in pigment granules in retinal pigment epithelium (RPE) cells, and (iv) altered morphology of Muller cells. DPC included both rods and cones. Their location and morphology depended on the stage of their displacement. These cells were usually oval or rounded in shape and were found either among the outer segments of other photoreceptor cells, having stalks extending into the outer nuclear layer, or were located in the subretinal space and had no stalk. A narrow space around the DPC stalks, indicating a change in the intercellular connection between photoreceptor cells and Muller cells, was observed. Furthermore, the Muller cells related to DPC had shortened and markedly reduced microvilli. Two types of macrophages were found in the subretinal space of aged monkey retinas. One type was similar in morphology to RPE cells. Some of these cells were noticed detaching from RPE. Other types of macrophages were nonpigmented. The modifications in RPE were closely related to the changes in the associated neuroretina. The RPE cells in aged retina were devoid of microvilli or had a few thin microvilli. The pleomorphic pigment granules were dispersed throughout the cytoplasm. These cells varied in their size, shape, and surface features. These changes could significantly alter the retinal metabolic equilibrium and may be indicative of age related degenerative processes.     

A quantitative cytochemical study of determination for xylem-element formation in response to wounding in roots of Pisum sativum L.

Quantitative cytochemical studies of cortical parenchyma cells of roots of Pisum sativum in which the central vascular bundle is severed, showed esterase activity to be an early marker of the determination of cells to form a vascular bridge. Explantation, onto a basal culture medium, of wound segments taken from roots at different times after severing the stele showed the irreversibility of the esterase activity on removal from the inducing environment, so confirming this as a marker of cell determination. A general determination for the stele was shown to occur by 8–10 h after wounding, but information relating to tracheid secondary-cell-wall formation was not apparently available until 18–20 h after wounding. Determination appeared to occur well before mitosis. The timings of the differentiation steps indicate a simple diffusion model to explain the mechanism of arrival of the initiating molecules.     

A quantitative cytochemical method for phosphofructokinase in plant tissues

A quantitative cytochemical method for the demonstration of phosphofructokinase has been successfully applied to a range of plant tissues. The findings indicate that this enzyme system may be assayed as an indicator of glycolytic activity in plant cells, and furthermore tha the very high endogenous phosphoenolpyruvate concentrations may not be rate limiting in vivo.     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.     

Improved bromodeoxyuridine/DNA analysis by anti-BudR monoclonal antibody versus right angle light scatter

Summary Dynamic cell cycle analysis is based on the incorporation of labelled precursors into DNA. Although antibodies to BrdU are very useful for analysing in flow cells which synthesize DNA, this approach has two main limitations. First, the detection of low incorporating cells is often difficult; second, four parameter flow cytometry is not able to correlate cell cycle to any other cellular marker. We have developed a methodology that, employing an IgG_H+L as a second antibody and side scatter instead of propidium iodide fluorescence, allows a better discrimination of BudR⁺ cells. This approach allows the collection of an extra-fluorescent signal, and the analysis of specific cellular markers within the cell cycle.     

Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets

The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.     

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

Localization of Drug-Metabolizing Enzyme Activities to Blood-Brain Interfaces and Circumventricular Organs

Abstract: The brain, with the exception of the choroid plexuses and Circumventricular organs, is partially protected from the invasion of blood-borne chemicals by the specific morphological properties of the cerebral micro-vessels, namely, the tight junctions of the blood-brain barrier. Recently, several enzymes that are primarily involved in hepatic drug metabolism have been shown to exist in the brain, albeit at relatively low specific activities. In the present study, the hypothesis that these enzymes are located primarily at blood-brain interfaces, where they form an "enzymatic barrier," is tested. By using microdissection techniques or a gradient-centrifugation isolation procedure, the activities of seven drug-metabolizing enzymes in isolated microvessels, choroid plexuses, meningeal membranes, and tissue from three Circumventricular organs (the neural lobe of the hypophysis, pineal gland, and median eminence) were assayed. With two exceptions, the activities of these enzymes were higher in the three Circumventricular organs and cerebral microvessel than in the cortex. Very high membrane-bound epoxide hydrolase and UDP-glucuronosyltransferase activities (approaching those in liver) and somewhat high 7-benzoxyre-sorufin- O -dealkylase and NADPH-cytochrome P-450 reductase activities were determined in the choroid plexuses. The pia-arachnoid membranes, but not the dura matter, displayed drug-metabolizing enzyme activities, notably that of epoxide hydrolase: The drug-metabolizing enzymes located at these nonparenchymal sites may function to protect brain tissue from harmful compounds.