ALBERT

Notes: Summary We have reviewed the stages in teleost thyroid function and its regulation, from the initial biosynthesis of the TH to their eventual interaction with putative receptors. TH biosynthesis depends on an adequate plasma iodide level, determined partly by dietary iodide and partly by active branchial iodide uptake from the water, Pulse-injected radioiodide can be used to evaluate thyroidal iodide uptake, aspects of TH biosynthesis and TH thyroidal secretion. However, owing to variable plasma iodide levels, care is required in interpretating these parameters. TH biosynthesis, thyroglobulin properties and intrathyroidal secretion mechanisms have received limited recent attention. Histological indices of thyroid tissue changes, while useful in many situations, do not always correlate with more direct estimates of thyroidal secretion and can be misleading. Thyroid function is regulated by the hypothalamo-pituitary-thyroid axis, but neither the identities of the hypothalamic factors nor a reliable immunoassay for TSH have been established. Currently, activity of the hypothalamic-pituitary axis is usually determined by pituitary thyrotrope histological appearance or bioassay of pituitary TSH. Plasma free T4 feeds back at both the pituitary and hypothalamic levels and inhibits TSH release. Thyroidal T4 secretory activity is presumably adjusted to maintain a constant plasma T4level according to physiologic state. Plasma T4 is probably the most commonly used index of thyroidal status. However, (1) T4 is probably not the active form of TH, (2) the T4 plasma level may be influenced by the binding properties of plasma proteins, and (3) the T4 concentration alone makes no provision for the rate of T4 turnover in plasma. The most practical way to measure thyroidal T4SR is to determine plasma T4DR, and assuming steady-state conditions, equate it to T4SR. The T4DR is determined from kinetic studies employing*T4, which also enable estimates of sizes of vascular and extravascular T4 pools and their rates of exchange. Excretion of T4 or its derivatives in urine or bile can be determined also. A high proportion of T4 is enzymatically monodeiodinated in liver and other tissues, generating T3 for local (intracellular) and vascular systemic compartments. Bothin vivo andin vitro methods have been used to quantify T4 deiodinase activity, which is highly responsive to physiologic state and environmental variables. T3 production is inhibited by a moderate T3 excess indicating an autoregulatory system, whereby tissue T3 levels are maintained at a set-point appropriate for a particular physiologic state. The rate of T3 production provides an informative measure of thyroidal status in a given tissue. However, other pathways also contribute to the maintenance of T3 homeostasis at a particular set-point. These include the rate of T3 degradation to 3,3′-T2, the rate of T4 substrate diversion to rT3 (an inactive isomer) and by the excretion of parent compounds or conjugates in bile and urine. Potential losses across branchial or integumentary surfaces have yet to be evaluated. The most fundamental measure of thyroidal status is represented by the amount of T3 saturably bound to receptors/nucleus for the cell type of interest. This is estimated most accurately in double isotope studies in which T3 contributions from both vascular and intracellular compartments are evaluated. Less satisfactory but meaningful indices of T3 availability to receptor sites may be obtained from the plasma T3 (or free T3) level and from the tissue T3 level. The former is appropriate if the cell type in question obtains its T3 primarily from plasma; the latter should be measured if the cell type derives its T3 mainly through intracellular deiodinase activity. If the proportion of vascular T3/intracellular T3 bound to receptors is known, it may indicate the degree of receptor activation. However, even cytosolic T3 levels may not vary in proportion to nuclear T3 levels. Differences in thyroidal function between teleosts and homeotherms can be attributed to distinctive strategies in iodide economy and to fundamental differences in control of thyroidal status. Owing to more certain iodide availability (branchial iodide pump and plasma iodide-binding proteins), teleosts are probably more liberal in their iodide use and have less efficient mechanisms for recovery and retention of hormonal iodide than homeotherms. Also, primary control of teleost thyroidal function appears peripheral. It is the finely regulated conversion of T4 to T3 in tissues which may largely determine the T4 secretion rate. Thus, T4, as a prohormone, may be produced more to satisfy the substrate needs for T4 conversion rather than to drive T3 production. Because TH are mainly implicated in tissue- or cell-specific processes involved in development, growth and reproduction in teleosts, it may be advantageous for their thyroidal status to be determined locally through T4-to-T3 deiodination. In homeotherms, primary control is mainly central through the hypothalamic-pituitary axis, which regulates thyroidal secretion of T4 and significant amounts of T3. The level of T4 (free T4) is believed to drive the production of T3 in most peripheral tissues. Because TH are extensively involved in the systemically integrated adjustment of basal metabolic rate in homeotherms, it may have been advantageous to evolve a system leaning towards central control by the hypothalamus, the brain centre associated with thermoregulation.

Notes: Synopsis The effects of acid ((H2SO4) and aluminum AIKSO4 in acidified water on rainbow trout, Salmo gairdneri, olfactory organ were examined using scanning electron microscopy and electrophysiology. Exposure to pH 4.7 resulted in an increase in the number of mucus droplets over parts of the olfactory epithelium, primarily along the ridges of the secondary folds. The addition of aluminum (5.0, 9.5, 20.0 µmol · 1−1) at pH 4.7 resulted in loss of receptor cell cilia, irregularly shaped olfactory knobs, clumped microvilli and swellings on microridge cells. Electrical responses recorded from the olfactory nerve in response to the amino acid L-serine were similar to controls in fish exposed to acidified water. When fish were exposed to acidified water and aluminum the response was depressed. These morphological and electrophysiological changes could be used to indicate metal-induced stress in fish from natural ecosystems.

Notes: Abstract This paper describes the design, modeling, and experimental test results of a single crystal silicon micromechanical device developed to evaluate fracture and fatigue of silicon based micromechanical devices. The structure is a cantilever beam, 300 microns long, with a large silicon plate and gold inertial mass at the free end. Torquing and sensing electrodes extend over the plate, and with associated electronics, drive the structure at resonance. Fatigue crack propagation is measured by detecting the shift in the natural frequency caused by the extension of a preexisting crack introduced near the fixed end of the cantilever. Experimental data are presented demonstrating time-dependent crack growth in silicon. Crack extensions of 10 to 300 nm have been measured with a resolution of approximately 2.5 nm, and crack tip velocities as low as 2.1×10−14 m/s. It is postulated that static fatigue of the native surface silica layer is the mechanism for crack growth. The methodology established here is generic in concept, permitting sensitive measurement of crack growth in larger fatigue specimens as well.

Notes: A radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGFα) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N-terminal to an octapeptide which is cognate to the N-terminal cleavage sequence of TGFα. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell-associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane-bound prepro TGFα. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo- and endo-peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1-2% of total cell activity of these enzyme classes.

Notes: We have previously characterized the stimulation of HeLa cell surface peptidase activity directed toward a nonapeptide substrate in response to low fluences of ultraviolet irradiation [Brown et al. (1993): J Cell Biochem 51:102-115]. To explore the hypothesis that this comprised a global response to cell stress featuring the interruption of DNA synthesis, a variety of agents affecting macromolecular synthesis were applied to HeLa cell cultures. Actinomycin D, 5,6-dichloro-1 β-ribofuranosyl benzimadazole, mitomycin C, ultraviolet light, and cycloheximide at doses which inhibited cell growth, but fell short of increasing the proportion of cells which had lost cell membrane impermeability to trypan blue, resulted in the concentration dependent increase in both amino- and endo-peptidase activities of intact HeLa cell cultures. γ-Irradiation, despite inhibiting an increase in cell number over a 20-h observation period, had no effect on the expressed level of cell surface peptidase activity nor did the accumulation of cells in S or G2 phase by thymidine parasynchronization. Some of these agents were found to increase the proportion of cells in the culture undergoing apoptosis (programmed cell death), and a strong correlation was found between the extent of apoptosis and the degree of elevation in cell surface peptidase activity. Higher concentrations of perturbants in some instances increased the percentage of cells that were nonviable and an associated release of intracellular proteases overwhelmed the linear correlation with apoptotic cells. The present data do not distinguish between a homogeneous elevation of surface peptidase activity in all cells of treated cultures or the heterogeneous increase in only preapoptotic or apoptotic cells. Since sunburn of the skin increases both the occurrence of apoptotic keratinocytes (sunburn cells) in the affected epidermis and the release of membrane bound cell activators such as transforming growth factor α, it is suggested by way of extrapolation of these in vitro results, that the increase in cell surface proteolytic activity plays an integral part in the reparative responses of the epidermal cells in vivo.

Notes: Several forms of perturbation result in the release of bioactive molecules into the microenvironment of injured cells to mediate the inflammatory or reparative reactions which restore normal tissue structure and function. Amongst other products, ultraviolet irradiation (UV) causes the release of the growth factor TGFα from a variety of epithelial cell sources, apparently by a post-translational mechanism. Here we have explored the hypothesis that UV results in the activation of cell surface proteases which may then be capable of excising mature TGFα from its plasma membrane-bound precursor. Using a recently described, sensitive assay of peptidase activity tailored to the substrate requirements for cleavage of the scissile bonds in proTGFα, we have found that nonlethal fluences of UV ( 〈 12 Jm-2) to HeLa cell cultures are followed by large increases in cell surface proteolytic activities. Amongst these, endopeptidase activity produces a similar product profile from the nonapeptide substrate to that of human leukocyte elastase, an enzyme previously shown to be capable of releasing a bioactive, mature form of TGFα from its cell-bound precursor. However, in addition to this candidate “TGFase” activity, cell surface aminopeptidase activity was also very significantly increased. The increase in the two classes of peptidase function differed in the timing of their responses. Aminopeptidase activation occurred immediately following UV, peaking after some 15-20 h, whereas the increase in endopeptidase activity lagged 6 h behind, cresting after 20-24 h. No evidence for a role for aminopeptidase in the activation of the endopeptidase could be found. Also, there was no increase in the total proteolytic activity demonstratable in cell extracts following UV.Attempts to interrupt the UV peptidase activation by inhibiting protein synthesis with cycloheximide were unsuccessful; rather, the inhibitor itself caused an increase in both classes of peptidase activity during the first 20 h. Unlike the UV response, both the aminopeptidase and endopeptidase ectoactivities increased simultaneously within a few hours of introducing cycloheximide into the medium of unirradiated cultures. The cycloheximide induced activity peaked after 20 h. Interestingly, cycloheximide alone has previously been shown to potentiate TGFα release from a cell line producing its precursor constitutively.These data suggest that both UV and cycloheximide can initiate reactions in HeLa cells which result in ectopeptidase activation of a global nature. Since both agents result in rapid interruption of DNA synthesis, it is possible that this cell surface proteolytic response may be analogous to, or part of, the “mammalian genetic stress response.” The mechanism of the activation of the cell surface proteases appears to be post-translational, perhaps part of a proteolytic cascade originating from perturbed macromolecular synthesis. © 1993 Wiley-Liss, Inc.