Chemistry of loteprednol etabonate and related steroids. II. Reactions at ring C and NMR structural studies of the resulting compounds

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.     

Cloning of cDNA and genomic structure of the mouse gamma-glutamyl transpeptidase-encoding gene

We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse gamma-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exon and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.     

Cloning of mouse c-ros renal cDNA, its role in development and relationship to extracellular matrix glycoproteins

Renal organogenesis ensues following reciprocal interactions between the uninduced metanephric mesenchyme and the ureteric bud. Conceivably, the presence of ligands or growth factors on a given cell type, and expression of receptors, including receptor proto-oncogenes, on the other cell type of different lineage would facilitate such epithelial-mesenchymal interactions. During these interactions, other macromolecules, such as extracellular matrix (ECM) proteins, present at the epithelial-mesenchymal surface, also play a role in the kidney morphogenesis. In this study the proto-oncogene, c-ros, was cloned and sequenced; its role in the metanephric development was examined, and correlated with the changes in the expression of ECM proteins. The mouse c-ros renal cDNA, belonging to phosphotyrosine kinase (PTK) receptor family, had a translation product of 2340 amino acids. The extracellular domain had 32 N-linked glycosylation sites and 30 cysteine residues. The transmembrane segment had a hydrophobicity approaching approximately 3.5. Multiple phosphorylation sites, typical of a PTK catalytic unit, were present in the cytoplasmic domain. The 3' noncoding region did not contain any A(U)nA mRNA instability motifs. The c-ros mRNA was highly expressed on the ureteric bud branches and their tips and on the developing glomeruli. Competitive RT-PCR analyses revealed the c-ros expression was the highest at 13th day of gestation, and it declined to very low levels during the neonatal period. Exposure of metanephric kidneys to c-ros antisense-oligonucleotide, derived from the PTK domain, caused dysmorphogenesis of the kidney and loss of c-ros expression on the ureteric bud branches. Concomitant with the reduced c-ros gene expression, a decreased expression of ECM glycoproteins, in particular the proteoglycans, was observed. These findings suggest that the c-ros plays a role in the metanephric development, and its effects may be modulated by the ECM macromolecules present at the epithelial-mesenchymal interface.     

mda-7 (IL-24) Inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling

Despite therapeutic interventions including surgery, chemotherapy and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound anti-proliferative and cytotoxic effects in a variety of tumor cells, but not in non-transformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cells. Ad.mda-7 reduced the proliferation of rodent and human glioma cells in MTT assays and in colony formation assays. The anti-proliferative effects of Admda-7 were enhanced by radiation in a greater than additive fashion. In vitro, this cellular change correlated with enhanced cell numbers in G1/G0 and G2/M phases of the cell cycle, implying Ad.mda-7 radiosensitizes tumor cells in a cell cycle-independent manner. The radiosensitizing effects were not observed in cultures of non-transformed primary astrocytes. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. Ad.mda-7 enhanced p38 and ERK1/2 activity but did not alter JNK or Akt activity. Irradiation of cells expressing MDA-7 suppressed ERK1/2 activity and dramatically enhanced JNK1/2 activity without altering either Akt or p38 activity. Inhibition of JNK1/2, but not p38, signaling abolished the radiosensitizing properties of MDA-7. Inhibition of neither ERK1/2 nor PI3K signaling enhanced the anti-proliferative effects of Ad.mda-7, whereas combined inhibition of both pathways enhanced cell killing, suggesting that ERK and PI3K signaling can be protective against MDA-7 lethality.     

Effects of intraventricular and epicardial application of adenosine on electrical activity of medullary PGL neurons

The effects of intraventricular injection and epicardial application of adenosine on spontaneous electrical activity of nucleus paragigantocellularis lateralis (PGL) neurons in rostral ventrolateral medulla (RVLM) were examined in 35 anesthetized rats with sinoaortic denervation and vagotomy. The results obtained were as follows: (1) The spontaneous discharge of 121 PGL neurons (mean discharge rate: 22.5 +/- 1.9 spikes/s) were recorded in 35 rats. (2) In response to intraventricular injection of adenosine (0.5 mumol/kg), mean arterial pressure (MAP) was initially increased by 1.7 +/- 0.2 kPa(P < 0.001) and subsequently decreased by 4.6 +/- 0.5 kPa(P < 0.001), while the heart rate (HR) was decreased by 126.5 +/- 12.3 bpm (P < 0.001). Of 35 PGL spontaneous discharge units responsive to intraventricular injection of adenosine, 30 showed an average increase from 21.9 +/- 2.6 to 29.2 +/- 3.4 spikes/s (P < 0.001), 3 with no change, while 2 with a decrease. (3) Following epicardial application of 20 mmol/L adenosine, the BP and HR were not significantly changed, while the spontaneous discharge of 22 PGL neurons were increased from 18.8 +/- 1.9 to 26.9 +/- 2.8 spikes/s (P < 0.001), and that of 3 neurons was not changed. (4) The excitatory response of PGL neurons to intraventricular injection or epicardial application of adenosine was completely inhibited by pretreatment with selective adenosine A1-receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 500 micrograms/kg). (5) Following epicardial application of phenol or bilateral stellate ganglionectomy, adenosine failed to affect the activity of PGL neurons. The results obtained indicate that adenosine may stimulate cardiac sympathetic afferents through adenosine A1-receptor, thereby resulting in the activation of PGL neurons.     

Bile acids of Fel Ursi

The first direct separation of Fel Ursi using octadecyl silica (ODS) open column chromatography has been accomplished and provided to provide several pure, free and conjugated bile acids, the structures of which were substantiated by spectroscopic methods.     

Changes in steady-state conformational equilibrium resulting from cytoplasmic mutations of the Na,K-ATPase alpha-subunit

Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.