Biomedical subjects

J D Kieffer

Publications and source records attributed to J D Kieffer.

At least 19 recordsLinked to original sources

PubMed2005

Juvenile atlantic and shortnose sturgeons (family: Acipenseridae) have different hematological responses to acute environmental hypoxia.

Experiments were conducted to determine the behavioral and physiological responses to acute hypoxic challenges in Atlantic (Acipenser oxyrinchus) and shortnose (Acipenser brevirostrum) sturgeons. We measured the ventilatory rate following a 45-mmHg hypoxic challenge, as well as a variety of hematological parameters, including O2 transport and hormonal, ionic, and metabolic variables, following a 1-h exposure to either 75- or 30-mmHg hypoxic challenges. Compared to fish in normoxic conditions (Pwo2 150 mmHg), juveniles of both species increased their ventilatory rate by approximately 40% when exposed to a 1-h challenge at 45 mmHg Pwo2. Hematological variables (e.g., hematocrit, hemoglobin, and Na+ and Cl- levels) did not change substantially following a 1-h challenge at 75 mmHg Pwo2. Conversely, a severe hypoxic challenge of 30 mmHg caused changes in several hematological variables (e.g., whole blood glucose and plasma cortisol and lactate levels). Most of these hematological parameters returned to prehypoxic levels within 2 h. Severe environmental hypoxia elicited the same basic pattern of response in both species; however, maximal plasma lactate levels were higher in Atlantic sturgeons, and maximal cortisol levels were higher in shortnose sturgeons. Whether these species differences are related to dissimilar hypoxia-tolerance, ecological, and/or endocrinological characteristics between these two species is not entirely clear.

Analysis of Variance↗

PubMed2004

Effects of metal mining effluent on Atlantic salmon (Salmo salar) and slimy sculpin (Cottus cognatus): using artificial streams to assess existing effects and predict future consequences.

In the summer of 2000, the effects of metal mine discharge on fish growth and exercise performance were assessed at a Zn-Pb-Cu mine in New Brunswick, Canada. Juvenile Atlantic salmon (Salmo salar) were exposed to 0%, 20%, and 80% treated metal mine effluent in a mobile, fish-only artificial stream system. Fish were fed commercial salmon pellets throughout the study. Young-of-the-year slimy sculpins (Cottus cognatus) were exposed to the same treatments in a multitrophic level, modular artificial stream system or mesocosm, in which the fish were dependent on seeded algae and invertebrates for nutrition. Treatment concentrations were chosen to represent existing discharge dilutions (80%) and a scenario of reduced effluent discharge (20%) as predicted upon mine closure (scheduled for 2008). Al, Ba, B, Fe, Mn, Sr, Tl, Ti, and Zn increased in a concentration-dependent fashion across the three treatments. Salmon body burdens of Ba, Cd, Li, Cu, Mn, Se, Sr, and Zn were increased in the 80% treatment, while Tl increased across all treatment levels. Mortalities and depressions in growth in both fish species paralleled treatment concentrations (80%>20%>0%). Salmon liver weight was significantly greater in fish exposed to 20% and 80% effluent in a concentration-dependent fashion. Exercise performance in fish, as assessed by the ability to recover from forced exercise, showed little effect of treatment. The contamination of the receiving environment by mine discharges has led to loss of fish, making it impossible to study the system in situ. However, the use of the artificial stream systems enabled us to assess effects of present conditions on fish, as well as the potential impacts of mine reclamation. The 20% discharge predicted following mine reclamation is potentially favourable for the reinstitution of native fishes into the system.

Animals↗

PubMed2003

The physiological response of diploid and triploid brook trout to exhaustive exercise.

Using triploidy as an experimental model, we examined whether cell size limits the post-exercise recovery process in fish. Because triploids generally possess larger cells, which could affect many physiological and biochemical processes, we hypothesized that triploids would take longer to recover from exhaustive exercise compared to diploids. To test this, we measured plasma lactate, glucose and osmolality, and white muscle energy stores (glycogen, phosphocreatine and ATP) and lactate before and immediately following exhaustive exercise and during recovery at 2 and 4 h post-exercise. In addition, oxygen consumption and ammonia excretion rates were determined before and after exhaustive exercise. Overall, diploid and triploid brook trout showed similar metabolic responses exercise, but plasma osmolality, white muscle lactate, white muscle ATP and post-exercise oxygen consumption rates recovered earlier in triploids compared to diploids. The results of this study suggest that the characteristic larger cell size of triploidy does not limit the physiological response to, or recovery from, exhaustive exercise.

Adenosine Triphosphate↗

PubMed2001

Neutrophils, monocytes, and dendritic cells express the same specialized form of PSGL-1 as do skin-homing memory T cells: cutaneous lymphocyte antigen.

Memory T cells in inflamed skin express the cutaneous lymphocyte-associated antigen (CLA), a glycosylated epitope defined by the mAb HECA-452. We previously reported that on T cells, CLA occurs almost exclusively on the protein backbone of P-selectin glycoprotein ligand-1 (PSGL-1). T cells exhibiting the CLA isoform of PSGL-1 can tether and roll on both E- and P-selectin, while T cells expressing PSGL-1 without the CLA epitope do not bind E-selectin, though they may bind P-selectin. We show here that circulating neutrophils and monocytes, and cultured blood dendritic cells, also express CLA almost entirely as an isoform of PSGL-1. These cells all tether and roll on both E- and P-selectin. A chimeric fusion protein incorporating the 19 N-terminal amino acids of mature PSGL-1 exhibited HECA-452 immunoreactivity and supported rolling of CHO cells expressing either E- or P-selectin. These findings indicate a site for the CLA modification within the distal tip of PSGL-1, previously shown to be critical for P-selectin binding and to mediate some, but not all, of the E-selectin binding of PSGL-1. We hypothesize that the types of circulating leukocytes discussed above all use CLA/PSGL-1 to tether and roll on E- and P-selectin along the vascular endothelium.

Animals↗

PubMed2001

Inflammatory skin disease in K14/p40 transgenic mice: evidence for interleukin-12-like activities of p40.

The proinflammatory cytokine interleukin-12, a p35/p40 heterodimer, is produced by resident cells in skin and has been implicated as a pathogenetic factor in T-cell-mediated skin diseases. Secretion of heterodimeric interleukin-12 is always accompanied by production of p40 monomer and p40/p40 homodimer. To investigate the possible in vivo role of p40 per se, we generated mice that constitutively express monomeric and homodimeric p40 in basal keratinocytes. These mice spontaneously developed an eczematous skin disease that was characterized by hyperkeratosis, focal epidermal spongiosis, and a mixed inflammatory infiltrate composed of T cells (CD4+), macrophages, eosinophils, mast cells, and few neutrophils. Fluorescence-activated cell sorter analysis of transgenic epidermal cell suspensions revealed induction of major histocompatibility complex class II molecules on keratinocytes and a 2-3-fold increase in the content of Langerhans cells. Cytokines produced by these activated epidermal cells include interleukin-1alpha and tumor necrosis factor alpha. The skin disease in K14/p40 mice was similar to that of littermate mice that received injections of interleukin-12, suggesting overlapping in vivo functional properties. As induction of interferon-gamma is a major function of interleukin-12, we tested the in vitro ability of transgenic p40 to induce interferon-gamma. In contrast to interleukin-12, transgenic p40 did not stimulate interferon-gamma secretion by cultured splenocytes. We conclude that transgenic p40 and interleukin-12 are equally capable of initiating cutaneous inflammation. Despite these in vivo similarities, there is a clear functional difference between interleukin-12 and transgenic p40 in vitro, suggesting that interferon-gamma is not a major factor contributing to interleukin-12-like activities of transgenic p40.

Animals↗

PubMed2001

Tonsillar B cells do not express PSGL-1, but a significant fraction displays the cutaneous lymphocyte antigen and exhibits effective E- and P-selectin ligand activity.

Skin-homing T cells are defined by the expression of the cutaneous lymphocyte-associated antigen (CLA) which enables the cells to selectively bind to vascular endothelial E-selectin close to sites of cutaneous inflammation, an initial step in the effective extravasation from blood into the inflamed tissue. Essentially all CLA on T cells decorates the backbone of the P-selectin glycoprotein ligand-1 (PSGL-1). In this study we show that human peripheral blood B cells (PBBC) and tonsillar B cells (TBC) do not display PSGL-1 in fluorescence-activated cell sorter analysis using different murine monoclonal antibodies and polyclonal rabbit anti-PSGL-1 antiserum. A significant population of TBC, however, expresses a HECA-452-reactive epitope. These cells represent nonactivated IgM(+)/IgG(-) mature B lymphocytes. Up to 50% of the TBC in a given preparation strongly bind to E- and up to 79% to P-selectin. The shear stress resistance in a parallel-plate flow chamber system was high. Neuraminidase treatment of TBC totally and O-sialoglycoprotein endopeptidase partially diminished HECA-452 reactivity and reduced E- but not P-selectin ligand activities. Mocarhagin had no effect in the assays. The data suggest a different ligand for P-selectin and a distinct glycoprotein carrier for the E-selectin ligand as compared to T cells or other leukocytes. Adhesion to P-selectin, however, still required sulfation of the ligand for function. Western blots of TBC cell lysates detected a >240-kD HECA-452-reactive material that was resistant to reducing conditions. Anti-PSGL-1 did not reveal immunoreactive material in these cell lysates. B cell activation did neither significantly change HECA positivity nor induce PSGL-1 expression. Cultured, activated TBC, however, maintained expression of the integrin alpha4beta7. Human peripheral blood B cells had similar cell surface characteristics to TBC. Our observations suggest that several adhesion molecules may be involved in B cell homing which include CLA, the P-selectin ligand, and structures such as alpha4beta7.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2001

Juvenile sturgeon exhibit reduced physiological responses to exercise.

Experiments were conducted to determine the physiological responses to exercise of Atlantic sturgeon (Acipenser oxyrhynchus) and shortnose sturgeon (A. brevirostrum). We measured the rates of oxygen consumption and ammonia excretion in both species and a variety of physiological parameters in both muscle (e.g. lactate, glycogen, pyruvate, glucose and phosphocreatine concentrations) and blood (e.g. osmolality and lactate concentration) in juvenile shortnose sturgeon following 5 min of exhaustive exercise. In both species, oxygen consumption and ammonia excretion rates increased approximately twofold following exhaustive exercise. Post-exercise oxygen consumption rates decreased to control levels within 30 min in both sturgeon species, but post-exercise ammonia excretion rates remained high in Atlantic sturgeon throughout the 4 h experiment. Resting muscle energy metabolite levels in shortnose sturgeon were similar to those of other fish species, but the levels decreased only slightly following the exercise period and recovery occurred within an hour. Under resting conditions, muscle lactate levels were low (<1 micromol g(-1)) but they increased to approximately 6 micromol g(-1) after exercise, returning to control levels within 6 h. Unlike similarly stressed teleost fish, such as the rainbow trout, plasma lactate levels did not increase substantially and returned to resting levels within 2 h. Plasma osmolality was not significantly affected by exercise in shortnose sturgeon. Taken together, these results suggest that shortnose and Atlantic sturgeon do not exhibit the physiological responses to exhaustive exercise typical of other fish species. They may possess behavioural or endocrinological mechanisms that differ from those of other fishes and that lead to a reduced ability to respond physiologically to exhaustive exercise.

Ammonia↗

PubMed2000

Limits to exhaustive exercise in fish.

Exercise to exhaustion leads to severe metabolic, acid-base and ionic changes in fish. It has been shown that several abiotic and biotic factors can limit burst exercise performance and the recovery process in fish. This article reviews the importance of body size, temperature, fasting/starvation and training on the ability of fish to perform and recover from exhaustive exercise. It is concluded that the constraints placed on a fish prior to and following exercise reflects the large intra-specific variability in the physiological response to exercise in fish.

Animals↗

PubMed1999

Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance.

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452-reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin-deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.

Animals↗

PubMed1997

Cutaneous lymphocyte antigen is a specialized form of PSGL-1 expressed on skin-homing T cells.

T cells play a pathogenic role in many inflammatory and certain malignant skin diseases, including psoriasis, atopic and allergic contact dermatitis, and cutaneous T-cell lymphoma. Memory T cells that infiltrate the skin express a unique skin-homing receptor called cutaneous lymphocyte-associated antigen (CLA), a carbohydrate epitope that facilitates the targeting of T cells to inflamed skin. CLA is defined by both its reactivity with a unique monoclonal antibody, HECA-452, and its activity as a ligand for E-selectin, but the structure of the protein component of CLA has not previously been defined. Here we report that CLA is an inducible carbohydrate modification of P-selectin glycoprotein ligand-1 (PSGL-1), a known surface glycoprotein that is expressed constitutively on all human peripheral-blood T cells. Cultured peripheral-blood T cells can be differentiated into CLA-bearing cells, which bind both E-selectin and P-selectin, or CLA-negative cells, which bind P-selectin but do not bind E-selectin, suggesting that there is independent regulation of selectin-binding phenotypes. We propose that differential post-translational modification of a single cell-surface receptor, PSGL-1, mediated by fucosyltransferase VII, serves as a mechanism for regulating tissue-specific homing of memory T cells.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1995

Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro.

The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

Actins↗

PubMed1995

Inflammatory skin disease in transgenic mice that express high levels of interleukin 1 alpha in basal epidermis.

Resting epidermal keratinocytes contain large amounts of interleukin 1 (IL-1), but the function of this cytokine in the skin remains unclear. To further define the role of IL-1 in cutaneous biology, we have generated two lines of transgenic mice (TgIL-1.1 and TgIL-1.2) which overexpress IL-1 alpha in basal keratinocytes. There was high-level tissue-specific expression of transgene mRNA and protein and large quantities of IL-1 alpha were liberated into the circulation from epidermis in both lines. TgIL-1.1 mice, which had the highest level of transgene expression, developed a spontaneous skin disease characterized by hair loss, scaling, and focal inflammatory skin lesions. Histologically, nonlesional skin of these animals was characterized by hyperkeratosis and a dermal mononuclear cell infiltrate of macrophage/monocyte lineage. Inflammatory lesions were marked by a mixed cellular infiltrate, acanthosis, and, in some cases, parakeratosis. These findings confirm the concept of IL-1 as a primary cytokine, release of which is able to initiate and localize an inflammatory reaction. Furthermore, these mice provide the first definitive evidence that inflammatory mediators can be released from the epidermis to enter the systemic circulation and thereby influence, in a paracrine or endocrine fashion, a wide variety of other cell types.

Animals↗

PubMed1995

The effects of blood CO2 reaction rates on CO2 removal from muscle in exercised trout.

The purpose of this study was to examine the relative importance of some of the factors potentially limiting carbon dioxide (CO2) removal from the white muscle in the rainbow trout following exhaustive exercise. Inhibition of red blood cell carbonic anhydrase (CA) activity by acetazolamide (Az) infusion following exercise caused a large increase in both arterial and muscle PCO2 indicating retention of CO2 in the animal. Despite the increase in muscle PCO2, there was no significant decrease in muscle pH in Az-treated fish, probably due to the relatively high buffer capacity of white muscle. The presence of CA in the plasma had a transient, significant effect on blood PCO2 and pH, but no significant effect on muscle PCO2 or pH. Thus, CA treatment transiently enhances CO2 removal from the animal following exercise, but the extracellular catalysis of the CO2 reactions in the plasma does not appear to have a large impact on CO2 removal from the tissues.

Acetazolamide↗

PubMed1995

Effect of 3,5,3'-Triiodothyronine (T3) administration on dio1 gene expression and T3 metabolism in normal and type 1 deiodinase-deficient mice.

The type 1 deiodinase (D1) catalyzes the monodeiodination of T4 to produce T3, the active thyroid hormone. In the C3H mouse, hepatic D1 and the dio1 messenger RNA (mRNA) are only 10% that in the C57 strain, the common phenotype. Low activity cosegregated with a series of five GCT repeats located in the 5'-flanking region of the C3H dio1 gene that impaired C3H promoter potency and provided a partial explanation for the lower D1. The present studies were performed to search for additional explanations for low D1 activity in C3H mice. Previous studies have shown that T3 up-regulates the dio1 gene. Therefore, loss of the capacity to respond to endogenous T3 is a possible additional cause of the lower D1 levels in the C3H mice. The hepatic C3H dio1 mRNA increases 10- to 20 fold after T3 administration. The t3 effect occurs at a transplantation level and T3 does not alter the dio1 mRNA half-life. Despite the transcriptional response to T3, no functional thyroid response elements were identified in the 1.5-kilobase 5'-flanking region of either the C57 or C3H dio1 gene. After the same dose of exogenous T3, both dio1 mRNA and D1 of the C3H mouse respond to a greater extent than those of the C57 strain. This can be explained in part by the reduction in T3 clearance due to the lower D1 levels in C3H mice in which higher concentrations of circulating T3 are maintained. The decrease in serum T3 levels and T3 production observed in fasting and systemic illness in both human and experimental animals has been attributed in part to a decrease in hepatic D1. In contrast, despite markedly lower hepatic and renal D1 levels, serum T3 concentrations remain normal in C3H mice. The present studies suggest that the absence of stress-induced hypothalamic-pituitary suppression that allows T4 production to be maintained together with the reduced clearance of T3 and T4 via inner ring deiodination compensate for the D1 deficiency.

Animals↗

PubMed1993

Physiological and genetic analyses of inbred mouse strains with a type I iodothyronine 5' deiodinase deficiency.

Inbred mouse strains differ in their capacity to deiodinate iododioxin and iodothyronines, with strains segregating into high or low activity groups. Metabolism of iododioxin occurs via the type I iodothyronine 5'deiodinase (5'DI), one of two enzymes that metabolize thyroxine (T4) to 3,5,3'-triiodothyronine (T3). Recombinant inbred strains derived from crosses between high and low activity strains exhibit segregation characteristic of a single allele difference. Hepatic and renal 5'DI mRNA in a high (C57BL/6J) and low (C3H/HeJ) strain paralleled enzyme activity and concentration, in agreement with a recent report. 5'DI-deficient mice had twofold higher serum free T4 but normal free T3 and thyrotropin. Brown adipose tissue 5'DII was invariant between the two strains. Southern analyses using a 5'DI probe identified a restriction fragment length variant that segregated with 5'DI activity in 33 of 35 recombinant inbred strains derived from four different pairs of high and low activity parental strains. Recombination frequencies using previously mapped loci allowed assignment of the 5'DI gene to mouse chromosome 4 and identified its approximate chromosomal position. We propose the symbol Dio1 to denote the mouse 5'DI gene. Conserved linkage between this segment of mouse chromosome 4 and human HSA1p predicts this location for human Dio1.

Animals↗

PubMed1992

Transcriptional and posttranscriptional mechanisms in uncoupling protein mRNA response to cold.

Three mechanisms account for the rapid elevation and maintenance of uncoupling protein (UCP) mRNA levels in cold-exposed rats, namely, an increase in the rate of transcription initiation, an increase in the fraction of nascent UCP transcripts undergoing elongation, and stabilization of the mature UCP mRNA. The second mechanism precedes and outlasts the increase in the rate of UCP gene transcription, which is brisk but short lived. After 48 h of cold exposure, mature UCP mRNA levels are maintained elevated solely on the basis of stabilization, since the levels of both transcription initiation and fifth intron-containing transcripts (precursors) have returned to basal. Results in hypothyroid rats given 3,5,3'-triiodothyronine (T3) and in dispersed brown adipocytes show that T3 is involved both in the increase in UCP mRNA precursor level and stabilization of mature UCP mRNA. These mechanisms are rapidly reversed when the rats are returned to thermoneutrality. These coordinated transcriptional and post-transcriptional mechanisms modulating UCP gene expression ensure a rapid increase in the concentration of UCP and prevent further accumulation of the protein as physiologically adequate levels are attained.

Animals↗

PubMed1992

Adenosine 3',5'-monophosphate and thyroid hormone control of uncoupling protein messenger ribonucleic acid in freshly dispersed brown adipocytes.

We intend to develop in vitro model systems to study the hormonal regulation of uncoupling protein (UCP) and its role in brown adipose tissue (BAT) thermogenesis. We report here that UCP mRNA responses to adrenergic and thyroid hormone manipulations in freshly dispersed, mature brown adipocytes mimic in vivo observations. Studies were performed in brown adipocytes obtained from interscapular brown fat of euthyroid or hypothyroid rats. The tissue was dispersed with collagenase, and cells were isolated by floatation over 4% BSA. UCP mRNA in these cells is 2-3 times more abundant than that in the whole tissue, indicating a selection of cells expressing the gene. In cells from euthyroid rats, UCP mRNA is maximally elevated within 2 h of exposure to 1 microM forskolin and 50 ng T3/ml (77 nM total, 0.43 nM free). T3 significantly enhances the effect of forskolin. In the absence of stimulation, UCP mRNA rapidly disappears from euthyroid brown adipocytes, and this can be prevented with the addition of T3 by a mechanism not requiring on-going transcription. In cells from hypothyroid rats, forskolin or isoproterenol plus phenylephrine fail to stimulate UCP mRNA, but within 3 h of exposure to T3, cells recover full responsiveness. As in vivo, a high extracellular concentration of T3 is required for maximal responsiveness of UCP mRNA to cAMP, while T4 can restore responsiveness in physiological concentrations (40 pM). This effect of T4 is prevented by iopanoic acid, a compound that blocks the type II T4 5'-deiodinase. In conclusion, 1) freshly dispersed brown adipocytes retain all of the properties concerning UCP regulation by thyroid hormone and sympathetic nervous system described for brown fat in vivo; 2) the observations made in vivo, thus, represent direct action of norepinephrine and thyroid hormone on these cells; 3) as in vivo, T4 is a better source of intracellular T3 than extracellular T3 for brown adipocytes; hence, the in vivo findings are the result of the cell biology of 5'-deiodinase type II rather than dynamic factors inherent to the in vivo condition; 4) stabilization of mature UCP mRNA by T3 is an important mechanism to maintain the levels of this mRNA elevated under sustained stimulation; and 5) dispersed brown adipocytes and UCP gene products constitute a powerful model to study interactions between the sympathetic nervous system and thyroid hormone at a cellular or molecular level.

Adipose Tissue, Brown↗

PubMed1992

Substitution of cysteine for selenocysteine in type I iodothyronine deiodinase reduces the catalytic efficiency of the protein but enhances its translation.

Type I iodothyronine 5' deiodinase (5'DI) contains selenocysteine, encoded by a UGA codon, and this amino acid is essential for maximum catalytic efficiency in this enzyme. We recently showed that translation of UGA as selenocysteine in this protein requires a specific sequence of about 250 nucleotides in the 3' untranslated region of the messenger RNA. Translation of a 5'DI cysteine mutant does not require the 3' untranslated region. To examine both the efficiency of UGA codon recognition and the relative catalytic efficiency of selenocysteine vs. cysteine in 5'DI, we used bromoacetyl 125I-T3 labeling to quantitate transiently expressed selenocysteine (wild type) and cysteine containing type I iodothyronine deiodinases in transfected COS-7 and JEG-3 cell lines. Kinetic analyses of the same cell sonicates were performed to determine the apparent maximum velocity and Michaelis-Menten constant values for reverse T3 5' deiodination. COS-7 cells express the cysteine mutant protein at about 20-fold and JEG-3 cells about 400-fold higher levels than the selenoenzyme. However, in both cell types, the apparent catalytic constant values were at least 100-fold higher for the wild-type enzyme, compared with the cysteine mutant. These results indicate that cell lines differ markedly in their capacity to translate UGA-containing messenger RNAs. The much higher catalytic constant values for the selenium-containing enzyme illustrate the biochemical advantage of this element as compared with sulfur in the catalysis of iodothyronine deiodination.

Catalysis↗