Expression of tumor necrosis factor-stimulated gene-6 in the rat ovary in response to an ovulatory dose of gonadotropin.

Current evidence supports the hypothesis that the biochemical events of mammalian ovulation are analogous to an acute inflammatory reaction. This study reveals that tumor necrosis factor-stimulated gene-6 (TSG-6), which encodes a member of the superfamily of hyaluronan-binding proteins that is specifically translated in inflammatory reactions, is expressed in ovarian follicles that have been induced to ovulate. Immature Wistar rats were primed with 10 IU equine CG s.c.; and 48 h later, the 12-h ovulatory process was initiated by 10 IU human CG (hCG), s.c.. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after the primed animals were injected with hCG. The RNA extracts were used for RT-PCR differential display of amplified complementary DNAs (cDNAs) that represented gene expression in the stimulated ovarian tissue. Northern analysis of one of the differentially amplified cDNAs confirmed that it was part of a gene that was substantially up-regulated at 4-8 h after the ovaries had been stimulated by hCG. Subcloning and sequence analysis revealed that the cDNA matched the gene for TSG-6. In situ hybridization indicated that the TSG-6 messenger RNA was primarily located in the cumulus mass and the antral granulosa cells of large ovarian follicles. In conclusion, the data show that expression of TSG-6 is an integral part of the cascade of inflammatory-like changes that occur in an ovulatory follicle in response to a trophic hormone that couples with luteinizing hormone/hCG receptors.

Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells.

FSH stimulates in ovarian granulosa cells diverse, differentiation-dependent responses that implicate activation of specific cellular signaling cascades. In these studies three kinases were investigated to determine their relationship to FSH, cAMP, and A kinase signaling: protein kinase B (PKB/Akt), serum and glucocorticoid-induced kinase (Sgk), and p38 mitogen-activated protein kinase (p38MAPK). The phosphorylation (activation) of these kinases was analyzed by using selective agonists/inhibitors: forskolin/H89 for cAMP-dependent protein kinase (A kinase), insulin-like growth factor I (IGF-I)/LY294002 and wortmannin for phosphatidylinositol-dependent kinase (PI3-K), and phorbol myristate (PMA)/GF109203X for diacylglycerol and Ca++-dependent kinases (C kinases). An inhibitor (PD98059) of MEK1, which regulates extracellular regulated kinases (ERKs), and SB203580, which inhibits p38MAPK, were also used. In addition, we analyzed the expression of the recently described, cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFI and GEFII) that impact Ras-related GTPases and Raf kinases, known regulators of various protein kinase cascades. We provide evidence that FSH, forskolin, and 8-bromo-cAMP stimulate phosphorylation of PKB by mechanisms involving PI3-K (LY294002/wortmannin sensitive) not A kinase (H89 insensitive), a pattern of response mimicking that of IGF-I. In contrast, FSH induction and phosphorylation of Sgk protein requires A kinase (H89 sensitive) but also involves PI3-K (LY294002 sensitive) as well as p38MAPK (SB203580 sensitive) pathways. PMA (C kinase) abolished FSH-mediated (but not IGF-I-mediated) phosphorylation of PKB at a step(s) upstream of PI3-K and independent of A kinase. Lastly, FSH-mediated phosphorylation of p38MAPK is negatively affected by A kinase and PI3-K, suggesting that it may be downstream of specific members of the cAMP-GEF/Rap/Raf pathway. We propose that cAMP activation of A kinase is obligatory for transcription of Sgk in granulosa cells whereas cAMP (IGF-I-like)-mediated phosphorylation (activation) of PKB and Sgk (via PI3-K), as well as p38MAPK, involves other cellular events. These results provide new and exciting evidence that cAMP acts in granulosa cells by A kinase-dependent and -independent mechanisms, each of which controls specific kinase cascades.

Hammerhead ribozyme cleavage of apolipoprotein B mRNA generates a truncated protein.

Target substrate-specific hammerhead ribozyme cleaves the specific mRNA and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To modulate apoB gene expression, we designed hammerhead ribozymes targeted at AUA(6665) and GUA(6679) of apoB mRNA, designated RB16 and RB15, respectively, and investigated their effects on apoB mRNA in HepG2 cells. The results demonstrated that RB15 and RB16 ribozyme RNAs cleaved apoB RNA efficiently in vitro. Both ribozymes, RB15 and RB16, were used to construct recombinant adenoviral vectors, designated AvRB15 and AvRB16, respectively, for in vivo gene transfer. HepG2 cells were infected with 2 x 10(5) plaque-forming units of AvRB15 for 5, 10, 15, and 24 h. An RNase protection assay showed that the expression of the RB15 transcript was time-dependent; it increased approximately 300-fold from 5 to 24 h. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site of apoB mRNA was confirmed by sequencing. Importantly, the levels of apoB mRNA in HepG2 cells decreased approximately 80% after AvRB15 infection. Pulse/chase experiments on HepG2 cells treated with AvRB15 and AvRB16 demonstrated that ribozyme cleavage produced a truncated protein that was secreted at a density of 1. 063-1.210 g/ml. The cleavage activity of RB15 on apoB mRNA was more efficient than that of RB16. Moreover, pulse/chase experiments in HepG2 cells treated with AvRB15 revealed that most of the truncated apoB protein was degraded intracellularly. We conclude that hammerhead ribozyme targeted at GUA(6679) of apoB mRNA cleaves apoB mRNA, results in decreased apoB mRNA levels, and generates a truncated apoB of the expected size in vivo. Thus, the therapeutic application of ribozyme in regulating apoB production holds promise.

Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1). structure-function relationships of RNA editing and dimerization.

APOBEC1 is the catalytic subunit of an enzyme complex that mediates apolipoprotein (apo) B mRNA editing. It dimerizes in vitro and requires complementation factor(s) for its editing activity. We have performed a systematic analysis of the structure-functional relationship of APOBEC1 by targeted mutagenesis of various sequence motifs within the protein. Using in vitro RNA editing assay, we found that basic amino acid clusters at the amino-terminal region R15R16R17 and R33K34, are essential for apoB mRNA editing. Mutation of R15R16R17 to K15K16K17 and mutation of R33K34 simultaneously to A33A34 almost completely abolished in vitro editing activity. The carboxy-terminal region of APOBEC1 contains a leucine-rich motif. Deletion analysis of this region indicates that residues 181 to 210 are important for in vitro apoB mRNA editing. Single amino acid substitutions demonstrate that L182, I185, and L189 are important residues required for normal editing function. Furthermore, the double mutant P190A/P191A also lost >90% of editing activity which suggests that a beta turn in this region of the molecule may be essential for proper functioning of APOBEC1. It was suggested that dimerization of APOBEC1 creates an active structure for deamination of apoB mRNA. When we examined the dimerization potential of truncated APOBEC1s using both amino and carboxy termini deletion mutants, we found that amino-terminal deletions up to residue A117 did not impair dimerization activity whereas carboxy-terminal deletions showed diminished dimerization. The systematic and extensive mutagenesis experiments in this study provide information on the role of various sequence motifs identified in APOBEC1 in enzyme catalysis and dimerization.

Two approaches to clinical evaluation.

The authors describe two methods of clinical evaluation which, when combined, give faculty a more objective evaluation of clinical performance. One method examines student performance throughout the semester; the other evaluates the student during an actual performance examination. This combination of methods gives the instructor an opportunity to observe the students' clinical performance from two different perspectives and to assign the grade for the student's performance in the practicum setting.

Pulmonary tuberculosis: contributions of radiology in diagnosis and treatment.

This review records the important place that radiologic examinations have had in the diagnosis and treatment of pulmonary tuberculosis in the past nine decades. Very soon after the discovery of x-rays it became apparent that chest roentgenograms would be a help in identifying pulmonary lesions. As the quality of the examinations improved, the benefits became significantly better. Earlier lesions were diagnosed. Follow-up studies after medical or surgical treatment were increasingly helpful. Newly developed techniques allowed demonstration of extrapulmonary complications. Radiologic monitoring of collapse therapy played a critical role in the successes achieved. Even after the medical conquest of active tuberculous lesions by drug therapy, identification of new lesions and follow-up of treated patients remains a significant role for radiology. Control of this once fatal and widely feared disease depends on continued early recognition and appropriate treatment.