Cross-linking of T3 (CD3) with T4 (CD4) enhances the proliferation of resting T lymphocytes

Monoclonal antibodies reactive with defined T lymphocyte surface antigens were covalently coupled to protein A-Sepharose beads using the bifunctional imidoester, dimethyl pimelimidate. Sepharose-immobilized antibody reactive with T3 induced the proliferation of resting T lymphocytes in the presence of either recombinant interleukin 2 or phorbol myristate acetate. When monoclonal antibodies reactive with T3 and T4 were coupled to the same Sepharose bead (hereafter designated Sepharose (T3:T4)), proliferation was enhanced an average of three-fold. Similarly prepared Sepharose beads coupled to anti-T3 and anti-T8 also enhanced proliferation over that observed with anti-T3 alone. Sepharose (T3:T4) similarly increased the proliferation of T4+ lymphocytes and a T4+ clone but failed to enhance the proliferation of T8+ lymphocytes. The increased proliferation of T4+ lymphocytes resulted from a preferential activation of the T4+2H4- helper population over the T4+2H4+ suppressor-inducer population. The enhanced proliferation induced by Sepharose (T3:T4) could be completely inhibited by soluble anti-T4. These results suggest that perturbation of T3 may be a minimal signal for T cell activation and that the assembly of a multimeric complex including T3 and T4 may be required for optimal T cell activation.     

Purification of cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein(CTBP) which regulates nuclear T3 translocation

The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein(CTBP) was purified from rat kidney using Mono Q-Sepharose, Red sepharose and T3 affinity chromatography. CTBP which was partially purified by Red Sepharose column chromatography was adsorbed to T3 affinity column in the presence of 50 uM NADPH. The CTBP was eluted from the gel with the buffer which did not contain NADPH. One molecule of the purified CTBP(58 kDa) bound one molecule of T3 with 2.44 x 10(9) M-1 of affinity constant. The purified CTBP was activated not only by NADPH but also by NADP in the presence of dithiothreitol. The NADPH-activated form did not transfer T3 to nuclei, whereas NADP transformed the NADPH-activated CTBP to active form which was able to transfer T3 to nuclei. These results suggested that CTBP-dependent transport of T3 to nucleus is controlled by NADPH and NADP.     

Delayed equilibrium of pituitary triiodothyronine (T3) following an acute T3 administration.

To reconcile the knowledge on tissue T3 concentration with cellular metabolism regulatory mechanism of thyroid hormone secretion, the pattern of the change of tissue T3 concentration following an acute administration of T3 was studied in mice. Basal T3 concentration in serum, liver, brain and pituitary was 61, 173, 198 and 1630ng/100g, respectively. After 0.5 mug T3 dose, T3 concentration in serum and liver reached the maximum level 1 to 3 hrs following the administration and decreased exponetially thereafter, thus, maintaining almost constant tissue/plasma T3 ratio. In contrast, T3 increase in brain or pituitary was far delayed, not until 7 to 12 hrs following T3 injection, and then decreased parallel to that in serum. Furthermore, the magnitude of increase in pituitary T3 was limited when compared to that in liver. Thus, tissue/plasma T3 ratio in pituitary decreased markedly after the dose of T3. This finding suggests the possibility that there is blood-brain barrier or blood-tissue barrier for the transport of T3 in pituitary or brain, resulting in delayed equilibrium with that in serum. These results may also explain the delay of inhibition of TRH-induced TSH release after single dose of T3 as recently reported by Azizi et al. (1975).     

The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits.

Clonally distributed (clonotypic) antigen receptors on human T lymphocytes (alpha and beta chains) are associated with three invariable T3 polypeptide chains (T3 gamma, delta and epsilon), together forming the T3/T cell receptor complex. Monoclonal antibodies prepared against the two 20-kd T3 polypeptide chains demonstrated that T3-delta and T3-epsilon are distinct polypeptide chains. Only one monoclonal antibody (anti-T3-delta chain) reacted with the T cell surface as judged by indirect immunofluorescence, and by its mitogenicity for quiescent peripheral blood lymphocytes. Immunohistological staining and immunoprecipitation experiments showed that both the T3-delta and T3-epsilon chains are T cell-specific. As seen with the anti-alpha/beta chain reagent WT-31, anti-T3-delta chain monoclonal antibodies stained medullary thymocytes more intensely than cortical thymocytes, whereas the difference between the staining of cortical and medullary thymocytes was generally not apparent with anti-T3-epsilon chain antibodies. Because of this specificity and their ability to react with both the denatured and the native forms of each polypeptide chain, these new monoclonal reagents will be useful tools in studies of the biosynthesis and cell surface expression of the T3/T cell receptor complex during normal and malignant thymic differentiation.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.     

Thermodynamics of triple helix formation: spectrophotometric studies on the d(A)10.2d(T)10 and d(C+3T4C+3).d(G3A4G3).d(C3T4C3) triple helices.

We have stabilized the d(A)10.2d(T)10 and d(C+LT4C+3).d(G3A4G3).d(C3T4C3) triple helices with either NaCl or MgCl2 at pH 5.5. UV mixing curves demonstrate a 1:2 stoichiometry of purine to pyrimidine strands under the appropriate conditions of pH and ionic strength. Circular dichroic titrations suggest a possible sequence-independent spectral signature for triplex formation. Thermal denaturation profiles indicate the initial loss of the third strand followed by dissociation of the underlying duplex with increasing temperature. Depending on the base sequence and ionic conditions, the binding affinity of the third strand for the duplex at 25 degrees C is two to five orders of magnitude lower than that of the two strands forming the duplex. Thermodynamic parameters for triplex formation were determined for both sequences in the presence of 50 mM MgCl2 and/or 2.0 M NaCl. Hoogsteen base pairs are 0.22-0.64 kcal/mole less stable than Watson-Crick base pairs, depending on ionic conditions and base composition. C+.G and T.A Hoogsteen base pairs appear to have similar stability in the presence of Mg2+ ions at low pH.     

The effect of thyroxine (T4) administration on plasma levels of tri-iodothyronine (T3) and T4 in male white-tailed deer

Plasma concentrations of thyroxine (T4) and tri-iodothyronine (T3) were monitored for 6 hr in mature male white-tailed deer following i.m. administration of synthetic T4. Oral administration of 600, 800 and 1000 micrograms of T4 was mostly ineffectual in increasing plasma levels of T3 and T4. On the other hand i.m. administration of similar doses of T4 was followed by a higher degree of increase in T3 and lesser degree of increase in T4 levels. It appears that high doses of T4 (e.g. 1000 micrograms) are less effective in raising plasma values of T3 or T4 than low or intermediate doses.     

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.     

Pinctada fucata mantle gene 3 (PFMG3) promotes differentiation in mouse osteoblasts (MC3T3-E1)

Nacre is secreted from the mantle of pearl oysters. In vivo and in vitro experiments have demonstrated that water-soluble extracts of nacre stimulate osteoblast differentiation and matrix mineralization, but the component responsible for this activity is unclear. It was reported that Pinctada fucata mantle gene 3 (PFMG3) with an N-terminal signal peptide could be secreted into the nacre of P. fucata. Here we report that PFMG3 is specifically expressed at the outer fold of the mantle and could promote calcium carbonate crystal formation in vitro. Consistent with this observation, we found that matrix mineralization of MC3T3-E1 cells, a murine osteoblast cell line, is accelerated upon treatment with PFMG3. Intriguingly, we observed that alkaline phosphatase activity and cell viability are increased after treating MC3T3-E1 cell with PFMG3. mRNA levels of osteoblast-specific marker genes osteocalcin and osteopontin are also increased. We conclude that PFMG3 from the mantle of P. fucata promotes MC3T3-E1 osteoblast cell differentiation, matrix mineralization, and calcium carbonate deposition in vitro. Our findings provide new evidence that PFMG3 may be used as a potential therapeutic molecule for the treatment of osteoporosis.     

Selenium Deficiency Inhibits the Conversion of Thyroidal Thyroxine (T4) to Triiodothyronine (T3) in Chicken Thyroids

Selenium (Se) influences the metabolism of thyroid hormones in mammals. However, the role of Se deficiency in the regulation of thyroid hormones in chickens is not well known. In the present study, we examined the levels of thyroidal triiodothyronine (T₃), thyroidal thyroxine (T₄), free triiodothyronine, free thyroxine (FT₄), and thyroid-stimulating hormone in the serum and the mRNA expression levels of 25 selenoproteins in chicken thyroids. Then, principal component analysis (PCA) was performed to analyze the relationships between the selenoproteins. The results indicated that Se deficiency influenced the conversion of T₄ to T₃ and induced the accumulation of T₄ and FT₄. In addition, the mRNA expression levels of the selenoproteins were generally decreased by Se deficiency. The PCA showed that eight selenoproteins (deiodinase 1 (Dio1), Dio2, Dio3, thioredoxin reductase 2 (Txnrd2), selenoprotein i (Seli), selenoprotein u (Selu), glutathione peroxidase 1 (Gpx1), and Gpx2) have similar trends, which indicated that they may play similar roles in the metabolism of thyroid hormones. The results showed that Se deficiency inhibited the conversion of T₄ to T₃ and decreased the levels of the crucial metabolic enzymes of the thyroid hormones, Dio1, Dio2, and Dio3, in chickens. In addition, the decreased selenoproteins (Dio1, Dio2, Dio3, Txnrd2, Seli, Selu, Gpx1, and Gpx2) induced by Se deficiency may indirectly limit the conversion of T₄ to T₃ in chicken thyroids. The information presented in this study is helpful to understand the role of Se in the thyroid function of chickens.     

3T3-L1 preadipocyte differentiation and poly(ADP-ribose) synthetase

Differentiation of 3T3-L1 preadipocytes, induced by methyl-isobutylxanthine (MIX), dexamethasone (DEX), and insulin, results in cells with the morphological and biochemical characteristics of adipocytes. Following incubation of 3T3-L1 cells with MIX, DEX, and insulin, poly(ADP-ribose) synthetase activity decreased abruptly, remained low for several hours and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents. It was not observed when preparations were assayed in the presence of DNase I, indicating that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin. The change in synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. It appears to be differentiation specific since nondifferentiating 3T3-C2 control cells did not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.     

Characterization of canine triiodothyronine (T3) autoantibodies and their effect on total T3 in canine serum

A study of 3,5,3'-L-triiodothyronine autoantibody (T3 AA) in 18 dogs revealed an average apparent affinity constant for T3 of 2.24 +/- 1.78 X 10(10) M-1, an average T3 binding capacity of 639.3 +/- 666.5 ng/dl and a low thyroxine (T4) cross-reactivity (less than 1%) in all samples tested. A valid radioimmunoassay (RIA) procedure which involved heat treatment of samples for 1 hr at 70 degrees C and assay on Sephadex minicolumns was developed for measuring T3 in the presence of T3 AA. Total T3 was elevated (mean = 374.8 +/- 158.4 ng/dl) in samples in which T4 was in the normal canine range, but T3 was lower (mean = 96.1 +/- 63.3 ng/dl) in samples with T4 values in the hypothyroid range. For each sample the concentration of T3 not bound by T3 AA was calculated from the total T3 concentration, the affinity constant, and the binding capacity. In dogs with normal total T4 concentrations the average calculated T3 not bound by T3 AA was 147.2 +/- 144.4 ng/dl while in dogs with low total T4 the value was 15.7 +/- 26.3 ng/dl (normal canine range is 45-150 ng/dl). Canine samples containing T3 AA were compared to serum from three rabbits actively immunized against T3 to provide anti-T3 for commercial RIA. The rabbit T3-antisera had an average T3 affinity constant similar to those of the canine samples (1.57 X 10(10) M-1), but had average titer, T3 binding capacity, and total T3 values more than 10-fold higher. Our findings indicate that, in dogs with serum containing T3 AA and normal total T4 concentrations, a compensatory mechanism appears to exist to maintain non-T3 AA bound T3 within the range of normal total T3. This compensatory mechanism does not operate in those dogs with insufficient thyroid activity to maintain normal total T4 values.     

Thyrotropin (TSH) regulates triiodothyronine (T3) production in the unicellular Tetrahymena

The aim of the experiments was to study the regulation of triiodothyronine (T3) production in the unicellular Tetrahymena. Untreated and troph-hormone treated specimen were prepared and in different timepoints T3 content was measured and compared by immunocytochemical flow cytometry. 0.1 or 0.001 IU TSH in tryptone-yeast medium stimulated T3 synthesis at 10, 20, 30 min, but does not stimulate after 1 h. The overlapping gonadotropic hormone (GTH) also did it, however only at 10 min. In Losina salt solution (physiological for Tetrahymena) the effect was weaker, however outer amino acid source was not absolutely needed for the production of the hormone. The results show that the TSH regulation of thyroid hormone synthesis (storage, secretion) and troph-hormone overlap can be deduced to a unicellular level. This may allow the hypothesis that the endocrine mechanisms proved at a low level of phylogeny are preserved for the higher ranked organisms.     

Density-dependent inhibition of growth: Inhibitorydiffusible factors from 3T3- and rous sarcoma virus (RSV)-transformed 3T3 cells

We recently fractionated, from the culture medium of 3T3 cells, a thermolabile inhibitory diffusible factor (IDF_N) with a molecular weight of about 40,000 daltons, which decreased nucleic acids synthesis of stimulated target 3T3 cells. In the present publication the inhibitory activities of IDF_N (produced by 3T3 cells) and IDF_T (produced by RSV-transformed 3T3 [3T3 SRA/H] cells) on 3T3 and 3T3 SRA/H cells have been compared. The inhibitory activity of IDF_N decreased (by a mean of 57%) when it was tested on transformed instead of 3T3 cells. On the other hand IDF_T was able to decrease ¹⁴C-inosine incorporation in target 3T3 cells. However, the inhibitory activity of IDF_T decreased (by mean 50%) when tested on 3T3 SRA/H instead of 3T3 cells. Therefore, transformed cells produced an inhibitory factor but were less sensitive than 3T3 cells to its inhibitory activity. The inhibitory activity of IDF_T on 3T3 SRA/H cells was only 20% of the inhibitory activity of IDF_N on 3T3 cells. This appreciable difference is of particular interest, since it could explain the release of density-dependent inhibition of growth (DDI) in transformed 3T3 SRA/H cells. Furthermore, it provides more evidence for the hypothesis that, in 3T3 cells, DDI of growth is due to the release of an inhibitory molecule into the medium, and that IDF_N is in fact, the inhibitory molecule involved in this phenomenon.     

Human T lymphocytes and monocytes bear the same Leu-3(T4) antigen

An analysis of the cellular distribution, biosynthesis, and structure of the human T lymphocyte antigen Leu-3(T4) was performed. By using a sensitive ELISA as well as FACS analysis, relative quantities of the Leu-3(T4) antigen from whole cell lysates and from cell surfaces of six cell lines were determined. The T-T hybrid cell line 255.88, and the monocyte/macrophage cell line U937, proved to be high producers of the antigen and were chosen for additional investigation. The Leu-3(T4) antigens from the T lymphocyte cell line and the monocyte/macrophage cell were shown to be identical by SDS-PAGE. Leu-3(T4) was a polypeptide of 55,000 AMW under reducing conditions, and 63,000 AMW under nonreducing conditions. In the 255.88 cell line, a second band of 41,000 AMW was associated with the true Leu-3(T4) molecule. The 55,000 AMW Leu-3(T4) molecule was shown to possess a high mannose sugar side chain, and to contain few accessible tyrosine residues. These studies demonstrate that human T lymphocytes and monocytes produce and process similar molecules that react with the anti-Leu-3(T4) monoclonal antibody. They also characterize this important associative antigen recognition structure and suggest that cells other than the T lymphocyte may be targets for the retrovirus HTLV-III.