Differential regulation of the pocket domains of the retinoblastoma family proteins by the HPV16 E7 oncoprotein

The human papillomavirus E7 oncoprotein binds to the retinoblastoma (Rb) tumor suppressor protein, and the binding to Rb correlates with the oncogenic potential of E7. Recent studies from several laboratories indicated that the half-life of the Rb protein is reduced in cells that are stably transformed with E7, suggesting that E7 could induce the proteolytic degradation of Rb. To investigate whether the Rb degradation is a primary effect of E7 or a result of altered cell phenotype, we sought to develop assays that can distinguish between the two possibilities. Using recombinant adenovirus expressing the human papillomavirus type 16 E7 protein, we show that the expression of E7 leads to an increased rate of decay of the Rb protein. Moreover, Rb degradation immediately follows the expression of E7 suggesting that it is an early and primary effect. Consistent with a previous study, we observed that the E7-induced degradation of Rb can be blocked by the inhibitors of the 26S proteasome. We have also developed a transient transfection assay for the E7-induced degradation of Rb. Using this assay, we show that the pocket domain of Rb is necessary and sufficient for the E7-induced degradation. However, the proteolysis is relatively specific for Rb because the level of p107 or p130 was not significantly altered by the expression of E7. Thus, although E7 binds to all three members of the Rb family of proteins, the proteolysis is much more efficient in the case of Rb. In the transient transfection assays, adenovirus E1A and SV40 large T antigen failed to induce degradation of Rb, suggesting that the Rb degradation is a unique property of the E7 oncoprotein.     

Differential expression of rat brain bcl-2 family proteins in development and aging

1. The effects of YM-60828, a newly synthesized factor Xa inhibitor, were investigated to analyse the relationship between its antithrombotic effects and its prolongation of template bleeding time in rats. YM-60828 was compared with argatroban, heparin and dalteparin. All agents were intravenously administered as a bolus. 2. In ex vivo studies, YM-60828 and argatroban prolonged both prothrombin time and activated partial thromboplastin time in a dose-dependent manner, while heparin and dalteparin prolonged only activated partial thromboplastin time. 3. In a venous thrombosis model, all agents exerted antithrombotic effects in a dose-dependent manner. The ID50 values of YM-60828, argatroban, heparin and dalteparin were 0.0081 mg kg(-1), 0.011 mg kg(-1), 6.3 iu kg(-1) and 4.7 iu kg(-1), respectively. 4. In an arterio-venous shunt model, all agents exerted antithrombotic effects in a dose-dependent manner. The ID50 values of YM-60828, argatroban, heparin and dalteparin were 0.010 mg kg(-1), 0.011 mg kg(-1), 10 iu kg(-1) and 4.2 iu kg(-1), respectively. 5. In bleeding time studies, all agents prolonged template bleeding time in a dose-dependent manner. ED2 values, the doses causing a 2 fold prolongation of bleeding time in the saline group, of YM-60828, argatroban, heparin and dalteparin were 0.76 mg kg(-1), 0.081 mg kg(-1), 18 iu kg(-1) and 25 iu kg(-1), respectively. 6. The ratio (ED2/ID50) of YM-60828 was more than 30 fold greater than that of heparin and more than 10 fold greater than those of argatroban and dalteparin. 7. These data show that YM-60828 can exert its antithrombotic effects with little prolongation of bleeding time compared with the other currently used anticoagulant agents.     

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation

To determine the possibility of discriminating multi-sources in the brain by 3D vector magnetic field measurement of a magnetoencephalogram (MEG), measurements were made of magnetic fields produced by two current dipoles implanted in a spherical head model. The 3D vector magnetic field measurements were made by using a 3D second-order gradiometer connected to three rf-SQUIDs, which can detect magnetic field components perpendicular to and tangential to the scalp. The MEG distribution measuring the magnetic field perpendicular to the scalp was not helpful in estimating the location and number of sources because of the lack of a dipole pattern. By referring to the MEG distribution measuring the magnetic field distribution tangential to the scalp, however, two current sources could be clearly discriminated in a spherical head model. It was found that this MEG distribution measuring tangential to the scalp could provide information on new constraint conditions for the calculation of inverse problems with multi-sources. These results were also confirmed by measurement of the mixed somatosensory evoked fields elicited by simultaneous electric stimulation to the median nerve and the thumb.     

Differential expression of the TEF family of transcription factors in the murine placenta and during differentiation of primary human trophoblasts in vitro

Konjac (konnyaku) glucomannan was examined for its degradation in human intestines and fermentation products. The konjac glucomannan was degraded almost 100% by soluble enzymes in human feces to give 4-O-beta-D-mannopyranosyl-D-mannopyranose (beta-1,4-D-mannobiose), 4-O-beta-D-glucopyranosyl-D-glucopyranose (cellobiose), 4-O-beta-D-glucopyranosyl-D-mannopyranose, and small amounts of glucose and mannose. These three disaccharides were further degraded by a cell-associated enzyme(s) to glucose or mannose, or to both. Konjac glucomannan underwent fermentation by intestinal anaerobic bacteria and produced formic acid, acetic acid, propionic acid, and 1-butyric acid. These fatty acids were different in their proportions among test subjects, their total amounts ranging from 17.1% to 48.8% of the initial konjac glucomannan.     

Differential expression of the retinoblastoma gene family members pRb/p105, p107, and pRb2/p130 in lung cancer

The Rb (retinoblastoma) gene family is composed of three members: the RB gene (one of the best-studied tumor suppressor genes) and two related genes, p107 and pRb2/p130. These three proteins share many structural and functional features and play a fundamental role in growth control. Using immunocytochemical techniques, we evaluated a variety of lung tumor specimens for the expression of this family of proteins and compared protein expression with the histological grading of the tumors and with the expression of the proliferating cell nuclear antigen. These Rb family members displayed distinctive patterns when compared and contrasted using different parameters. The highest percentage of undetectable levels in all of the specimens examined and the tightest inverse correlation (P) with the histological grading and with proliferating cell nuclear antigen expression in the most aggressive tumor types were found for pRb2/p130, which may suggest an important role for this protein in the pathogenesis and progression of lung cancer.     

Differential expression and mutation of the ras family genes in human breast cancer

The expression of ras mRNA levels in 27 human sporadic breast cancer specimens was examined, and compared to the corresponding adjacent normal tissue using the RT-PCR technique. Eighteen out of the 27 specimens (67%) exhibited two- to four-fold increased expression of ras mRNA levels, compared to corresponding normal tissue. The rates of augmented mRNA expression were similar among the three ras genes. A statistically significant correlation of overexpression of ras genes in specimens classified as Stage I disease was observed, compared to tumors in a more advanced stage (II or III). The incidence of codon 12 point mutations of the K-ras gene in fresh tissue samples was also assessed in 61 human sporadic breast cancer cases. Point mutations were detected in four (6.5%) out of the 61 cases examined; no correlation was found with any clinicopathological parameter. This is the first report to our knowledge of the differential expression of the ras family genes in breast carcinoma. Our findings indicate that the aberrant expression of ras genes may be an initial event in breast cancer oncogenesis and that K-ras point mutations are rarely involved in the development of mammary neoplasias.     

Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system

In this review, we summarize the structure and function of the scavenger receptor family of proteins including class A (type I and II macrophage scavenger receptors, MARCO), class B (CD36, scavenger receptor class BI), mucinlike (CD68/macrosialin, dSR-CI) and endothelial (LOX-1) receptors. Two motifs have been identified as ligand-binding domains: a charged collagen structure of type I and II receptors, and an immunodominant domain of CD36. These structures can recognize a wide range of negatively charged macromolecules, including oxidized low-density lipoproteins, damaged or apoptotic cells, and pathogenic microorganisms. After binding, these ligands can be either internalized by endocytosis or phagocytosis, or remain at the cell surface and mediate adhesion or lipid transfer through caveolae. Under physiological conditions, scavenger receptors serve to scavenge or clean up cellular debris and other related materials, and they play a role in host defence. In pathological states, they mediate the recruitment, activation and transformation of macrophages and other cells which may be related to the development of atherosclerosis and to disorders caused by the accumulation of denatured materials, such as Alzheimer's disease.     

Differential expression of the phenol family of UDP-glucuronosyltransferases in hepatoma cell lines

Procedural side effects and complications of chemical peeling should be considered as distinct entities. They are best managed by careful preoperative screening and operative technique and attentive surgical follow-up in the first postoperative week.     

Differential expression of rat brain synaptic proteins in development and aging

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.     

Differential expression of heat shock proteins in normal and failing human hearts

OBJECTIVE: To predict spinal cord ischemia after endovascular stent graft repair of descending thoracic aortic aneurysms, temporary interruption of the intercostal arteries (including the aneurysm) was performed by placement of a novel retrievable stent graft (Retriever) in the aorta under evoked spinal cord potential monitoring. METHODS: From February 1995 to October 1997, endovascular stent graft repair of descending thoracic aortic aneurysms was performed in 49 patients after informed consent was obtained. In 16 patients with aneurysms located in the middle and distal segment of the descending aorta, the Retriever was placed temporarily before stent graft deployment. The Retriever consisted of two units of self-expanding zigzag stents connected in tandem with stainless steel struts. Each strut was collected in a bundle fixed to a pushing rod, and the stent framework was lined with an expanded polytetrafluoroethylene sheet. The Retriever was delivered beyond the aneurysm through a sheath and was retracted into the sheath 20 minutes later. A stent graft for permanent use was deployed in patients whose predeployment test results with the Retriever were favorable. Evoked spinal cord potential was monitored throughout placement of the Retriever and stent grafting until the next day. RESULTS: The Retriever was placed in 17 aneurysms in 16 patients. There were no changes in amplitude or latency of evoked spinal cord potential records obtained before or during Retriever placement. After withdrawal of the Retriever, all aneurysms were excluded from circulation immediately after permanent stent grafting. There were no changes in evoked spinal cord potential, nor were neurologic deficits seen after stent graft deployment in any patient. CONCLUSIONS: These results suggest that predeployment testing with the Retriever under evoked spinal cord potential monitoring is promising as a predictor of spinal cord ischemia in candidates for stent graft repair of thoracic aortic aneurysms.     

Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy

Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively. Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.     

Differential expression and localization of IGF-I and IGF binding proteins in inflamed rat colon

Recent studies indicate increased insulin-like growth factor I (IGF-I) expression and altered expression of IGF binding proteins (IGFBP) in the bowel during experimental colitis. This study analyzes the cellular sites of altered IGF-I and IGFBP-expression in large bowel of rats with experimental colitis. Colitis was induced by colonic instillation of 2, 4, 6-trinitrobenzenesulfonic (TNB) acid in ethanol. Animals were sacrificed at 7 days after induction of colitis. Cryostat sections of colon from TNB-treated and control rats were hybridized with 35S-labeled antisense probes for IGF-I, IGFBP-3, IGFBP-4 and IGFBP-5. IGF-I mRNA was up-regulated in lamina propria cells, submucosa and smooth muscle of inflamed colon. IGFBP-3 mRNA was localized to lamina propria and was down-regulated in inflamed colon. IGFBP-4 and IGFBP-5 mRNAs were both up-regulated in inflamed colon. IGFBP-4 mRNA was increased in lamina propria, submucosa and smooth muscle, whereas IGFBP-5 mRNA was increased in smooth muscle. Increased IGF-I expression in mesenchymal layers of colon during experimental colitis supports the hypothesis that IGF-I contributes to hyperplasia and fibrosis in response to inflammation. Altered expression of IGFBP-3, IGFBP-4 and IGFBP-5 in specific bowel layers during colitis suggests that they play a role in modulating IGF-I action.     

Differences in extracellular matrix proteins, epidermal growth and differentiation in discoid lupus erythematosus, lichen planus and the overlap syndrome

In this review, we focus on the heterogeneity of interstitial lung diseases detected in patients with collagen vascular diseases. By recognizing the heterogeneity of histopathology and comparing them with bronchoalveolar lavage fluid cell findings, we can understand profiles of lung inflammation and injuries and fibrosis in collagen vascular diseases. We focus on the significance of lung lymphocytosis in the lesions of patients with collagen vascular diseases, looking most closely at lesions in unusual interstitial pneumonia. The current understanding of immunopathogenesis and immunopathological findings is reviewed in the context of subsets of collagen vascular diseases.     

Differential expression and distribution of focal adhesion and cell adhesion molecules in rat hepatocyte differentiation

The present study has examined the distribution of axons of differing sizes in the optic pathway of the ground squirrel. Axon diameters were measured from electron micrographs at various locations across sections of the optic nerve and tract, and total distributions and numbers were estimated. In both the nerve and tract, roughly 1.2 million optic axons were present. The population of optic axons had a unimodal size distribution, peaking at 0.9 microm in diameter and having an extended tail toward larger diameters. Local axon diameter distributions in the optic tract indicated distinct (though partially overlapping) axon diameter classes, including one of fine sizes peaking at 0.8-0.9 microm, a second of medium sizes peaking around 1.7-1.8 microm, and a third composed of the larger fibers with diameters up to 4.8 microm. The fine-caliber axons were found at all locations in the tract, and were the only axons present immediately adjacent to the pia, while the medium- and coarse-caliber axons were found at deeper locations. Curiously, the larger axons were found primarily in the medial parts of the tract, where axons from the dorsal retina normally course. A similarly restricted distribution of the larger axons was observed in the dorsotemporal parts of the optic nerve, suggesting that this difference in the tract may relate to an asymmetric distribution of ganglion cells on the retina giving rise to these axons. Measurements of axonal size taken within the optic fiber layer in dorsal and ventral parts of the retina confirmed this asymmetry, consistent with previous demonstrations of soma size differences in the dorsal versus ventral retina. The partial segregation of axons by size in the optic tract of the ground squirrel then reflects both the asymmetric distribution of retinal ganglion cell classes and the chronotopic reordering of optic axons that occurs within the chiasmatic region.