Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.     

Association of immune parameters with clinical outcome in stage III colon cancer: results of Southwest Oncology Group Protocol 9009

Levamisole (LMS), utilized in the adjuvant treatment of patients with stage III colon cancer, is immunomodulatory. To determine whether alterations in immune parameters before, during and after 12 months of 5FU/LMS therapy correlate with disease-free survival, 38 patients enrolled on Southwest Oncology Group (SWOG) protocol 8899 received extensive lymphocyte phenotypic analysis prior to therapy and 3, 6, 12 and 15 months after treatment initiation. The median follow-up of patients is 41 months. Significant increases in the proportion and total number of CD56⁺ natural killer cells were seen, starting at 3 months and continuing until 15 months (P < 0.001). Increases in the total numbers of cells expressing CD25 (interleukin-2 receptor), VLA4 and the combinations of CD4: CD45RA and CD4:CDw29 were not evident during therapy but were seen at 15 months (P < 0.05: CD25, CD4:CDw29, CD4:CD45RA; P < 0.001: VLA4). Low levels of CD8⁺ cells prior to treatment initiation and after 3 months of therapy correlated with early relapse within the first year of 5FU/LMS treatment. Patients who have remained disease-free (n = 22, median follow-up 45 months) demonstrated increases in the total numbers of CD8⁺, CD25⁺, CD56⁺, VLA4⁺, CD4: CDw29 and CD4:CD45RA cells, primarily at 15 months. In contrast, patients who relapsed had decreased numbers of CD8⁺, CD4:CDw29, CD4: CD45RA and VLA4⁺ cells and minimal increases in CD56⁺ and CD25⁺ cells. Statistically significant differences between the late-relapse group and the group remaining disease-free were seen for CD25⁺, CD4: CD45RA and CD4:CDw29 cells at the 15-month assay time (P = 0.0276, P = 0.0349, P = 0.0178 respectively). In conclusion, multiple alterations in lymphocyte phenotype, with increases in the proportion and total number of cells involved in cell-mediated immune responses, were seen during and especially following completion of therapy with 5FU/LMS. Many of these changes are significantly associated with clinical outcome and may be useful for risk stratification of stage III colon cancer patients following completion of adjuvant therapy. Received: 9 July 1999 / Accepted: 11 August 1999     

HIV-1 Tat directly binds to NFkappaB enhancer sequence: role in viral and cellular gene expression

HIV-1 Tat protein reprograms cellular gene expression of infected as well as uninfected cells apart from its primary function of transactivating HIV-1 long terminal repeat (LTR) promoter by binding to a nascent RNA stem–loop structure known as the transactivator response region (TAR). Tat also induces chromatin remodeling of proviral LTR-mediated gene expression by recruiting histone acetyl transferases to the chromatin, which results in histone acetylation. Furthermore several studies have shown convincing evidence that Tat can transactivate HIV-1 gene expression in the absence of TAR, the molecular mechanism of which remains to be elucidated. Here we show a direct interaction of Tat with nuclear factor kappa B (NFκB) enhancer, a global regulatory sequence for many cellular genes both in vitro and in vivo. This interaction not only provides a novel molecular basis to explain TAR-independent transactivation in HIV-1, but also points toward the potential mechanism of Tat- mediated modulation of cellular genes.     

Novel Techniques for Weak Alignment of Proteins in Solution Using Chemical Tags Coordinating Lanthanide Ions

A molecule with an anisotropic magnetic susceptibility is spontaneously aligned in a static magnetic field. Alignment of such a molecule yields residual dipolar couplings and pseudocontact shifts. Lanthanide ions have recently been successfully used to provide an anisotropic magnetic susceptibility in target molecules either by replacing a calcium ion with a lanthanide ion in calcium-binding proteins or by attaching an EDTA derivative to a cysteine residue via a disulfide bond. Here we describe a novel enantiomerically pure EDTA derived tag that aligns stronger due to its shorter linker and does not suffer from stereochemical diversity upon lanthanide complexation. We observed residual (15)N,(1)H-dipolar couplings of up to 8 Hz at 800 MHz induced by a single alignment tensor from this tag.     

Developmental regulation of prostaglandin E2 synthase in porcine ductus arteriosus

The synthesis of PGE(2), the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE(2) synthases (PGES). The factors implicated in increased PGE(2) synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A(2) (cPLA(2)) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE(2) in the DA of NB were approximately 7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA(2) in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE(2) in fetal DA to levels approaching those of the NB; cPGES, cPLA(2), and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE(2) levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE(2) levels in the perinate.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

Improved growth and essential oil yield and quality in Foeniculum vulgare mill on mycorrhizal inoculation supplemented with P-fertilizer

Two arbuscular mycorrhizal (AM) fungi Glomus macrocarpum and Glomus fasciculatum significantly improved growth and essential oil concentration of Foeniculum vulgare Mill. However, AM inoculation of plants along with phosphorus fertilization significantly enhanced growth, P-uptake and essential oil content of plants compared to either of the components applied separately. Among the two fungal inoculants, G. fasciculatum registered the highest growth at both levels of phosphorus used with up to 78% increase in essential oil concentration of fennel seeds over non-mycorrhizal control. The essential oil characterization by gas liquid chromatography revealed that the level of anethol was significantly enhanced on mycorrhization.     

Artemisinins: activities and actions

Multidrug-resistant malaria caused by Plasmodium falciparum has severely limited treatment options over recent years. Artemisinins are still effective for treating uncomplicated as well as severe malaria, because resistance is not yet clinically apparent. This article reviews some clinically useful properties of artemisinins and how they might work.     

Mycobacterium tuberculosis and Escherichia coli nucleoside diphosphate kinases lack multifunctional activities to process uracil containing DNA

E. coli nucleoside diphosphate kinase (EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK (MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung(-) strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.