A new regulatory element that augments the Tax-dependent enhancer of human T-cell leukemia virus type 1 and cloning of cDNAs encoding its binding proteins

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) trans activates the 21-bp enhancer of HTLV-1. A sequence of more than two copies of the 21-bp enhancer is efficiently activated by Tax, but one copy is not activated extensively. Another sequence (TRE-2, positions -163 to -117) adjacent to the 21-bp enhancer in the long terminal repeat of HTLV-1 can enhance a single copy of the 21-bp enhancer activity in trans activation by Tax. This sequence contains motifs related to the Ets- and NF-kappa B-binding sequences, but mutations at these sites indicated that neither is responsive to cooperation with the 21-bp enhancer. A deletion mutation of TRE-2 identified 25 bases at positions -158 to -134 (TRE-2S) as an essential sequence, and TRE-2S was sufficient to give maximum cooperation with one copy of the 21-bp enhancer in trans activation by Tax protein. Using TRE-2S as a probe, we screened a cDNA library of HUT102 cells by the Southwestern (DNA-protein) procedure and isolated two cDNA clones, THP-1 and -2. These two clones encode TRE-2S-binding proteins, and they differ by only an extra 17 amino acids in THP-2. Both THP proteins contain five zinc finger motifs which are strikingly similar to those of the GLI family, an amplified gene product in glyoma cells. The binding site of THP-1 and -2 was GAACCACCCA in TRE-2S, which is highly homologous to the GLI-binding site. These results suggest that binding of THP to TRE-2S may be involved in cooperation with one copy of the 21-bp enhancer in responding to Tax trans activation.     

Redox modulation of selenium binding proteins by cadmium exposures in mice

The mechanism of the antagonistic behaviour of selenium (Se) against cadmium (Cd) toxicity is investigated. This study reports the distribution of Cd at the organ and subcellular level after chronic treatments. The possible role of the selenium binding proteins (SBP) during Cd exposure are also evaluated. Kidney concentrates more Cd than liver following 8 weeks of treatment. Simultaneous administration of Se reduced Cd accumulation in Kidney. This affect did not occur in liver. Among the subcellular fractions, the maximum concentrations of both of the elements were found in the cytosol. The overall uptake of 75Se was enhanced in the cytosol of kidney and liver of the Cd treated animals. These observations support a hypothesis that selenium is complexed with cadmium. The increase in the labeling of SBP as a result of Cd exposures may reflect a change in the conformation of the protein molecule. These proteins (SBP) contain a sequence motif, which may be an active redox centre. Also, Cd significantly reduced the glutathione level, thereby disrupting the thiol/disulfide balance. This in turn may affect the redox status of the proteins leading to a 75Se or 75Se-Cd complex with SBP.     

Enhanced cell proliferation and biosynthesis mediate improved wound repair in refed, caloric-restricted mice

Aged mice that have undergone long-term caloric-restriction (CR) have improved health and enhanced longevity in comparison to aged mice that are ad libitum-fed (AL). However, caloric-restriction does not benefit the impaired wound healing of aged mice. To test the hypothesis that CR mice have the capacity for enhanced wound repair, but require a short-term period of additional nutrient intake to show this advantage, we assessed wound healing in CR mice that had been refed (RF) an ad libitum diet for 4 weeks prior to wounding. Two strains of AL young (Y AL) (4-6 months), AL middle-aged (M AL) (15-17 months), and three different, matched cohorts of old mice (O) (30-33 months): O AL, O CR, and O RF were studied. Two full-thickness 4 mm diameter punch biopsy skin wounds were created on the dorsum of each mouse. Animals were sacrificed and wounds were harvested at 1,2,3,5, and 7 days post-wounding. Repair of wounds was slower in O AL and O CR mice compared to Y AL and M AL animals. In contrast, the O RF mice healed similarly to that of the Y AL and M AL mice, as assessed by measures of wound area and histologic criteria. O RF mice demonstrated enhanced synthesis of type I collagen mRNA in comparison to O AL and O CR mice. A greater number of endothelial cells and fibroblasts at the wound edge of the O RF mice exhibited replication in vivo as measured by uptake of BrdU. O RF mice had higher levels of insulin-like binding protein 3 (IGFBP-3). Furthermore, fibroblasts derived from the explant of the punch biopsy of O CR mouse skin revealed enhanced proliferation and contraction in vitro, in comparison to fibroblasts from the O AL mice. In conclusion, O RF mice demonstrate an enhanced capacity to undergo wound repair in comparison to O AL mice. This effect appears to be mediated, in part, by enhanced cell proliferation, contraction, and collagen biosynthesis. In addition, short-term refeeding induced an increase in the serum level of IGFBP-3, the major binding protein for IGF-1. These data confirm that cells from O CR animals have a preserved proliferative, biosynthetic, and contractile capacity, but that an adequate source of nutrients is necessary to demonstrate this advantage in wound healing.     

Histone deacetylase. A key enzyme for the binding of regulatory proteins to chromatin

Stone and urine composition were analysed in 75 men and 40 women with recurrent calcium oxalate stone disease (group R) and in 48 men and 19 women who had formed only one calcium-oxalate-containing stone (group S). Patients who had developed stones with a large fraction of calcium phosphate were significantly more frequent in group R than in group S. There was furthermore a higher excretion of calcium and higher calcium oxalate supersaturation levels in patients with stones containing more than 25% calcium phosphate. It was concluded from these observations that the calcium phosphate content of renal stones might be a useful factor in predicting the future course of the disease.     

Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2

We investigated the suitability of a lyophilized bovine hemoglobin (LBH) preparation containing various fractions of oxyhemoglobin (O2Hb), carboxyhemoglobin (COHb), and methemoglobin (MetHb) for quality assessment in multicomponent analysis (MCA) of hemoglobin derivatives. It was demonstrated that a stable preparation of these components after reconstitution yields a hemoglobin solution that is spectrophotometrically equivalent with a fresh bovine hemoglobin solution. The preparation was found to be stable for at least 1 year when it is kept at 2-8 degrees C and for 1 h after reconstitution. We determined the fractions of O2Hb, COHb, and MetHb of several LBH preparations, using the complete spectra of 480-650 nm with 2-nm intervals and absorptivities as determined for pure LBH solutions. A field trial involving various types of multiwavelength hemoglobin photometers showed the suitability of LBH as a quality-control material. Computer models of the various common multiwavelength hemoglobin photometers may be useful for establishing more accurate target values of LBH preparations for each type of photometer and for studying the importance of the influence of specific factors such as wavelength selection, absorptivity values, and interfering dyes.     

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding

To examine a role for the medullary nucleus paragigantocellularis (PGi) in mediation of the symptomatology of opioid withdrawal, bilateral electrical stimulation of the PGi was performed in conscious, unrestrained, opioid naive (nondependent) rats. A characteristic series of behaviors was elicited during each 30-min session of PGi stimulation. The profile of these behaviors resembled qualitatively, but was not quantitatively identical with those seen during precipitated withdrawal from opioid dependence. This behavioral syndrome has been termed, opioid withdrawal-like behavior. The opioid withdrawal-like behaviors were voltage-, but not frequency-, dependent. Tolerance to repeated stimulation of the PGi did not develop following a series of 30-min runs of stimulation over 3.5 h. Intracerebroventricular (i.c.v.) injections of the nonselective opioid antagonist, naloxone, significantly decreased (by 40-50%) the intensity of stimulation-induced behavioral responses, as did injections of either the mu-selective (beta-funaltrexamine, beta-FNA) or the delta-selective (naltrindole, NTI) opioid antagonists. In contrast, similar i.c.v. injections of the kappa-selective antagonist, nor-binaltorphimine (nor-BNI), did not block behavioral responses to PGi stimulation. The results indicate that activation of the PGi by electrical stimulation can elicit behaviors similar to those observed during opioid withdrawal. Endogenous opioids, acting through mu- and delta-, but not kappa-opioid receptors, participate in mediating opioid withdrawal-like behaviors induced by PGi stimulation.     

The physiological role of sterol regulatory element-binding protein-2 in cultured human cells

To clarify the role of the sterol regulatory element-binding protein-2 (SREBP-2), we established cell lines in which human SREBP-2(1-481) could be induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The range of IPTG-induced changes in SREBP-2(1-481) levels in '23-11' cells, one of these cell lines, was almost the same as that of sterol-induced changes in the levels of mature SREBP-2, indicating that IPTG was able to regulate the expression of SREBP-2(1-481) within the normal physiological range in this cell line. Sterols regulate the expression of the LDL receptor, HMG-CoA reductase, squalene synthase and fatty acid synthase in 23-11 cells as they also do in the parental cell line HeLa S3. IPTG increased mRNA levels of the LDL receptor and HMG-CoA reductase but not squalene synthase both in the presence or absence of excess sterols. Fatty acid synthase mRNA was increased 2 h after the IPTG addition in the absence of excess sterol (10% FBS), but was slightly increased 6 h after the IPTG addition in the presence of excess sterols. In the absence of excess sterols, both SREBP-2(1-481) and endogenous mature SREBP-2 exist in the nucleus. This suggests that an increased amount of SREBP-2 over the normal physiological range is required for the regulation of fatty acid synthase. IPTG increased both the surface binding of 125I-LDL and cholesterol biosynthesis from [14C]acetate significantly in a similar time course. In contrast, fatty acid biosynthesis from [14C]acetate was almost unchanged by IPTG during the same incubation period. These results suggest that physiological amounts of SREBP-2 play a key role in the regulation of cholesterol but not fatty acid metabolism.     

Excitotoxic brain lesion modifies binding to a USF binding site acting as a negative regulatory element in the Protease nexin-1 promoter

This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia+ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAC). The presence of the supernatant in the mixture partially protected the Ia+ LC from depletion by the steroid. The Ia+ LC frequency increasing activity was measured as the difference between the Ia+ LC frequency due to treatment with steroid mixed with supernatant and the Ia+ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia+ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm2, as compared with 486 (SD 68) cells/mm2 in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a > or = 10 KDa molecular weight cut-off membrane was capable of partially protecting Ia+ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia+ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia+ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia+ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia+ LC.