Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.     

Arterial-phase three-dimensional gadolinium magnetic resonance angiography of the renal arteries. Strategies for timing and contrast media injection: original investigation

RATIONALE AND OBJECTIVES: The authors review different imaging and contrast-media infusion strategies for arterial-phase three-dimensional (3D) gadolinium-enhanced magnetic resonance angiography (Gd-MRA). METHODS: The influence of physicochemical factors on the infusion of contrast media, including viscosity, flow rate, inline pressure, and cannula size, is assessed. The combination of manual or automated contrast-media administration with timing-dependent or -independent 3D Gd-MRA techniques is reviewed regarding the aspects of effectiveness, robustness, image quality, and costs. RESULTS: For effective bolus delivery with high flow rates, the type and temperature of the contrast media, the size of the cannula, and an immediate saline flush must be considered. Timing-dependent techniques based on a test bolus and using automated contrast-media infusion as well as timing independent techniques such as MR SmartPrep or multiphase 3D Gd-MRA by using a manual injection with a SmartSet tubing set, are all effective procedures for arterial phase 3D Gd-MRA. CONCLUSIONS: Manual contrast-media injection with a tubing set can be used for timing-independent MRA techniques. The multiphase 3D Gd-MRA approach seems to be favorable for different MR systems, robustness, and speed.     

Dermatomyositis

Dermatomyositis is a rare inflammatory myopathy with characteristic skin manifestations and muscular weakness. The disease can be categorized as adult idiopathic, juvenile, or amyopathic dermatomyositis as well as that associated with a connective tissue disease or a malignancy. Immunologic factors are most likely involved in the pathogenesis of the disease; however, genetic and environmental issues may also play important roles. Treatment with immunosuppressive agents has proved successful in the majority of patients, although significant morbidity still occurs.     

Fast fluid-attenuated inversion-recovery (FLAIR) MRI in the assessment of intraaxial brain tumors

This study demonstrates the value of a fast fluid-attenuated inversion-recovery (FLAIR) technique in the assessment of primary intraaxial brain tumors. Twenty-one patients with primary intraaxial brain tumors were examined by T2-weighted, proton-density-weighted fast spin echo, fast FLAIR, and contrast-enhanced T1-weighted spin echo using identical slice parameters. The images were evaluated using quantitative and qualitative criteria. Quantitative criteria were tumor-to-background and tumor-to-cerebrospinal fluid (CSF) contrast and contrast-to-noise ratio (CNR). The qualitative evaluation was performed as a multireader analysis concerning lesion detection, lesion delineation, and image artifacts. In the qualitative evaluation, all readers found the fast FLAIR to be superior to fast spin echo in the exact delineation of intraaxial brain tumors (P < .001) and the delineation of enhancing and nonenhancing tumor parts. Fast FLAIR was superior in the delineation of cortically located and small lesions but was limited in lesions adjacent to the ventricles. Fast FLAIR provided a significantly better tumor-to-CSF contrast and tumor-to-CSF CNR (P < .001). The tumor-to-background contrast and tumor-to-background CNR of the fast FLAIR images were lower than those of T2-weighted spin-echo images but higher than those of proton-density-weighted spin-echo images. FLAIR images had more image artifacts influencing the image interpretation in only two patients. Signal hyperintensities at the ventricular border were present in 92% of the patients. They are common findings in fast FLAIR and should be included into the image interpretation.     

The microsomal triglyceride transfer protein catalyzes the post-translational assembly of apolipoprotein B-100 very low density lipoprotein in McA-RH7777 cells

In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL. A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (+BFA) and chased (+BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (-BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.     

Blocking interleukin-6 activity with chimeric anti-IL6 monoclonal antibodies in multiple myeloma: effects on soluble IL6 receptor and soluble gp130

Interleukin-6 (IL6) plays a major role in the pathogenesis of multiple myeloma. In patients with monoclonal gammopathy serum levels of sIL6R have been found to be increased. The role of IL6 in the regulation of soluble receptors is still unclear. In a phase I/II study we treated 12 myeloma patients with high-affinity chimeric anti-IL6 monoclonal antibodies. This treatment resulted in a total in vivo blockage of IL6 activity and as a result we had an unique opportunity to gain insight into the possible regulation effects of IL6 on these soluble IL6 receptors. Pre-treatment sIL6R levels were elevated in 9 of the 12 patients; pre-treatment sgp130 levels were significantly increased in all patients. Total blockage of IL6 activity by the high-affinity cMab did not influence sIL6R in 10 of these 12 patients and sgp130 levels remained stable in all patients. Of the 2 patients whose sIL6R levels increased during therapy, one had progressive disease and the other developed an acute infection. We conclude that in most end-stage myeloma patients sIL6R and sgp130 serum levels are elevated, but that there is no relation between IL6 activity and sIL6R or sgp130 levels.     

Tunneled hemodialysis catheters: use of a silver-coated catheter for prevention of infection--a randomized study

OBJECTIVE: The purpose of this study was to determine the feasibility of high-resolution sonography for the detection of meniscal cysts and associated meniscal tears and for the differentiation of meniscal cysts from other masses at the knee joint. SUBJECTS AND METHODS: Fifty consecutive patients (51 knees) with a palpable mass at the knee joint were examined prospectively using a 7.5-MHz annular array transducer. Mass consistency and location and meniscal integrity were evaluated. Sonographic findings were correlated with surgery (46/51) and histopathology (15/51). Five patients did not undergo surgery. RESULTS: At surgery, 32 masses appeared to be meniscal cysts, whereas 19 were other types of masses. Sonographically, 31 of the 32 meniscal cysts were diagnosed correctly. Sonographic differentiation of the other types of masses from meniscal cysts could reliably be made in 17 of 19 cases; two masses were falsely interpreted as meniscal cysts. Sensitivity, specificity, and accuracy of sonography in the depiction of meniscal cysts were 97%, 86%, and 94%, respectively. The positive predictive value was 94% and the negative predictive value was 92%. Meniscal tears (31/46) and meniscal tears concomitant with meniscal cysts (26/32) were detected with an accuracy of 83% and 88%, respectively. CONCLUSION: Sonography is an accurate imaging technique for the detection of meniscal cysts and associated meniscal tears. Differentiation of meniscal cysts from other cystic and solid masses at the knee joint can be reliably made with sonography.     

Long-range and short-range oxidative damage to DNA: photoinduced damage to guanines in ethidium-DNA assemblies

Short-range and long-range photoreactions between ethidium and DNA have been characterized. While no DNA reaction is observed upon excitation into the visible absorption band of ethidium, higher-energy irradiation (313-340 nm) leads both to direct strand cleavage at the 5'-G of 5'-GG-3' doublets and to piperidine-sensitive lesions at guanine. This reactivity is not consistent with oxidation of guanine by either electron transfer or singlet oxygen as shown by comparison with reactions of a rhodium intercalator and methylene blue, respectively. By covalently tethering ethidium to one end of a DNA duplex, we demonstrate the presence of two distinct reactions, one short-range and the other long-range. The short-range reaction involves a covalent modification of guanine by ethidium, based upon HPLC analysis of the nucleoside products and studies with ethidium derivatives. The long-range reaction is entirely consistent with oxidation of guanine by DNA-mediated electron transfer. The yield of this electron-transfer reaction is not attenuated with distance; equal yields of guanine damage are observed at a proximal (17 A Et-GG separation) and distal (44 A Et-GG separation) site. These results are quite similar to those previously observed with a covalently tethered rhodium photooxidant and underscore the unique ability of the DNA base stack to facilitate long-range electron transfer so as to effect oxidative damage from a distance.     

Role of the C9 methyl group in rhodopsin activation: characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal

Activation of the visual pigment rhodopsin involves both steric and electrostatic interactions between the chromophore and opsin within the retinal-binding site. Removal of the C9 methyl group of 11-cis-retinal inhibits light-dependent activation of the G protein, transducin, suggesting a direct steric contact. More recently, we have shown that steric interactions lead to receptor activation when Gly121 in the middle of transmembrane helix 3 is replaced by larger hydrophobic residues. In order to understand in more detail the role of the C9 methyl group of retinal in the structure and function of rhodopsin, we first studied the properties of recombinant 9-dm-Rho (opsin reconstituted with 11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shifted lambdamax, increased hydroxylamine reactivity, and decreased ability to activate transducin. These properties are consistent with the hypothesis that the C9 methyl group is a crucial structural anchor for the correct docking of the chromophore in its binding site. Next, we investigated the possible interaction between Gly121 of opsin and the C9 methyl group of retinal by characterizing recombinant pigments produced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind the chromophore. However, the double mutant G121L/F261A bound 11-cis-9-demethylretinal to form a stable pigment with a lambdamax of 451 nm. When activity was assayed in membranes, the reduction in transducin activation by 9-dm-Rho caused by the lack of a C9 methyl group on the chromophore could be partially restored by replacing Gly121 with a bulky residue (leucine, isoleucine, or tryptophan). These results support a model of receptor activation that involves steric interaction between the C9 methyl group of the chromophore and the opsin in the vicinity of Gly121 on transmembrane helix 3.     

Central GABAA and GABAB receptor modulation of basal and stress-induced plasma interleukin-6 levels in mice

The development of control strategies for loiasis is of crucial importance in endemic areas and depends heavily on the accurate identification of occult-infected individuals. A polymerase chain reaction (PCR) and nested polymerase chain reaction (nested PCR) were developed and based on sequences of the repeat 3 region (15r3) of the gene encoding a Loa loa 15-kD protein. The assays was performed on 20 blood samples from occult-infected subjects and 30 from field-collected amicrofilaremic individuals. The size of the initial PCR product was 396 basepairs (bp). When this initial amplification using primers 15r3(1) and 15r3(2) was carried out for 30 cycles, the PCR products from three of the 20 occult-infected and five of the 30 amicrofilaremic individuals were visualized after electrophoresis by staining the gel with ethidium bromide. Subsequent Southern blotting and hybridization with the specific probe revealed hybridization in 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples but only after two days of exposure of the blot to the x-ray film. When the nested PCR was carried out (product size = 366 bp, primers 15r3(3) and 15r3(4)), 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples that were positive by Southern hybridization of the initial PCR products were strongly positive by staining with ethidium bromide. Qualitative Southern blotting of the nested PCR products using the same probe previously described confirmed the ethidium bromide staining results after a very short exposure time of 4 hr. These results demonstrate that the nested PCR amplification product is specific and that its sensitivity in detecting occult loiasis is 95%. This approach has significant promise for the screening of large human populations for active loiasis without the requirement for blotting and hybridization of the PCR products.