Separation of DNA binding domain from hormone and core histone binding domains by trypsin digestion of rat liver nuclear thyroid hormone receptor

Rat liver nuclear thyroid hormone receptor was subjected to limited trypsin digestion, and the tryptic fragment of the 3,5,3'-triiodo-L-thyronine (T3)-receptor complex was characterized. Rat liver nuclear thyroid hormone receptor is an asymmetrical protein with Stokes radius of 34 A, sedimentation coefficient of 3.4 S, and molecular weight of 49,000. A globular T3-receptor complex with Stokes radius of 22 A, sedimentation coefficient of 2.8 S, and molecular weight of 26,000 was obtained by tryptic digestion. This fragment had no DNA binding activity, whereas undigested receptor showed significant DNA binding activity. Addition of undigested receptor to the tryptic fragment did not restore DNA binding activity of digested receptor, nor did mixing inhibit DNA binding activity of undigested receptor complex. Undigested receptor bound to core histones, and this activity was stronger than with other proteins tested (H1 histone, cytochrome c, and ovalbumin). The tryptic fragment of receptor maintained core histone binding activity comparable to that of undigested receptor. The tryptic fragment had affinity for T3 comparable to undigested receptor as assessed by Scatchard analysis and the same rate for dissociation of [125I]T3 from receptor. The tryptic fragment of the T3-receptor complex was more stable than undigested receptor at 43 degrees C. Digestion of receptor unoccupied by T3 caused a significantly larger loss of T3 binding capacity than did digestion of T3-occupied receptor, suggesting a protective effect of T3 on a second trypsin-sensitive site on the receptor, which, when cut, destroys T3 binding activity.     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Expression of three forms of thyroid hormone receptor in human tissues

At least two thyroid hormone receptor (hTR) genes are present in humans, but the significance of this multiplicity is unknown. These receptors could have differences in tissue distribution or possess different functions. We studied the distribution and abundance of three hTR mRNAs (hTR beta, hTR alpha 1, and hTR alpha 2) by Northern blot analysis. Three mRNAs were expressed in all tissues examined. hTR beta was strongly expressed in brain and prostate predominantly as a 10.0-kilobase (kb) mRNA. This mRNA was also expressed in thyroid and was much less abundant in liver, kidney, placenta, tonsil, and spleen. hTR alpha 1 is represented by two mRNAs with sizes of 6.0 and 3.2 kb. The 6.0-kb mRNA was constantly less abundant than the 3.2-kb mRNA. hTR alpha 2 was detected as a single mRNA with a size of 3.2 kb, using a probe unique for this mRNA. Both hTR alpha 1 and hTR alpha 2 were strongly expressed in brain, prostate, and thyroid and much less in other tissues. The relative amounts of the three hTR mRNAs were roughly parallel in each tissue. It is of interest that none of these hTRs was abundant in liver, which is the major thyroid hormone-responsive organ. Another hTR may be present in liver.     

Extremity cooling for heat stress mitigation in military and occupational settings

Physical work, high ambient temperature and wearing protective clothing can elevate body temperature and cardiovascular strain sufficiently to degrade performance and induce heat-related illnesses. We have recently developed an Arm Immersion Cooling System (AICS) for use in military training environments and this paper will review literature supporting such an approach and provide details regarding its construction. Extremity cooling in cool or cold water can accelerate body (core temperature) cooling from 0.2 to 1.0 °C/10 min vs. control conditions, depending on the size/surface area of the extremity immersed. Arm immersion up to the elbow results in greater heat loss than hand- or foot-only immersion and may reduce cardiovascular strain by lowering heart rate by 10–25 beats/min and increase work tolerance time by up to 60%. The findings from studies in this paper support the use of AICS prototypes, which have been incorporated as part of the heat stress mitigation procedures employed in US Army Ranger Training and may have great application for sports and occupational use.     

Identification of PLOD2 as telopeptide lysyl hydroxylase,an important enzyme in fibrosis

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.     

Evidence for trichloroethylene bioactivation and adduct formation in the rat epididymis and efferent ducts

Recent studies indicate that trichloroethylene (TCE) may be a male reproductive toxicant. It is metabolized by conjugation with glutathione and cytochrome p450-dependent oxidation. Reactive metabolites produced along both pathways are capable of forming protein adducts and are thought to be involved in TCE-induced liver and kidney damage. Similarly, in situ bioactivation of TCE and subsequent binding of metabolites may be one mechanism by which TCE acts as a reproductive toxicant. Cysteine-conjugate beta-lyase (beta-lyase) bioactivates the TCE metabolite dichlorovinyl cysteine (DCVC) to a reactive intermediate that is capable of binding cellular macromolecules. In the present study, Western blot analysis indicated that the soluble form of beta-lyase, but not the mitochondrial form, was present in the epididymis and efferent ducts. Both forms of beta-lyase were detected in the kidney. When rats were dosed with DCVC, no protein adducts were detected in the epididymis or efferent ducts, although adducts were present in the proximal tubule of the kidney. Trichloroethylene can also be metabolized and form protein adducts through a cytochrome p450-mediated pathway. Western blot analysis detected the presence of cytochrome p450 2E1 (CYP2E1) in the efferent ducts. Immunoreactive proteins were localized to efferent duct and corpus epididymis epithelia. Metabolism of TCE was demonstrated in vitro using microsomes prepared from untreated rats. Metabolism was inhibited 77% when efferent duct microsomes were preincubated with an antibody to CYP2E1. Dichloroacetyl adducts were detected in epididymal and efferent duct microsomes exposed in vitro to TCE. Results from the present study indicate that the cytochrome p450-dependent formation of reactive intermediates and the subsequent covalent binding of cellular proteins may be involved in the male reproductive toxicity of TCE.     

Extracellular mitochondrial DNA and oxidatively damaged DNA in synovial fluid of patients with rheumatoid arthritis

We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS–polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplifiable mtDNA was detected in the plasma of 30 of 54 (56%) of patients with RA and in 6 of 22 (27%) of the healthy volunteers. The levels of mtDNA in the plasma and SF samples of patients with RA were significantly higher (P < 0.0001) than in the respective control samples. The presence of both mtDNA and 8-oxodG in SF was significantly correlated with the presence of rheumatoid factor in the patients with RA. Extracellular mtDNA and oxidized DNA were detected in the SF of the great majority of patients with RA, but were absent or present at low levels in the control SF. These findings indicate that endogenous nucleic acid compounds might participate in joint inflammation by activating immune cells in the joints to produce proinflammatory cytokines.     

CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves' disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto's thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.