Recombinant forms of Gerbich blood group antigens: expression and purification.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.     

Anaesthetic potency of inhalation agents is independent of membrane microviscosity

The decrease in membrane microviscosity of erythrocyte ghosts in the presence of clinically relevant concentrations of seven inhalation anaesthetic agents was studied using fluorescence polarization anisotropy of the membrane incorporated fluorescent probes 1,6-diphenyl- 1,3,5-hexatriene and 1-[4-trimethylammoniumphenyl]-6-phenyl-1,3,5- hexatriene. All anaesthetic agents produced a dose-dependent decrease in anisotropy of both probes, indicating decreased membrane microviscosity. The reduction in anisotropy measured at the minimum alveolar concentration (ED50) for anaesthesia was related inversely to the anaesthetic potency of the agent and was directly proportional to the hypothetical concentration of agent in the membrane calculated from lipid-water partition coefficients. These findings do not support the hypothesis that volatile anaesthetic agents act by increasing membrane microviscosity of the bulk lipid bilayer to produce anaesthesia.      

Potential problem with fluorescence polarization immunoassay cross-reactivity to vancomycin degradation product CDP-1: its detection in sera of renally impaired patients

During the development of a homogeneous immunoassay for the antibiotic vancomycin, we observed in certain patient samples a quantitation difference between the enzyme multiplied immunoassay technique (EMIT) method and the comparison method, fluorescence polarization immunoassay (FPIA). This prompted us to evaluate the integrity of vancomycin in samples from renally impaired patients. Since it has been reported in the scientific literature that vancomycin degrades into an antibiotically inactive crystalline degradation product (CDP-1) in vitro, we developed high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) methods to determine whether CDP-1 is present in patient sera. HPLC and LC/MS analysis on samples from renally impaired patients positively identified CDP-1 in fresh samples. Next, we tested the cross-reactivity of three currently available vancomycin immunoassays, radioimmunoassay (RIA) FPIA, and EMIT, to CDP-1 prepared in our laboratory. Our data suggest that CDP-1 is recognized by FPIA and RIA, both polyclonal antibody-based methods, but not by EMIT, which uses a monoclonal antibody.     

Surgical Management of Renal Cell Carcinoma With Inferior Vena Cava Tumor Thrombus

Background. The optimal management of patients with renal cell carcinoma with inferior vena cava tumor thrombus remains unresolved. Traditional approaches have included resection with or without the use of cardiopulmonary bypass. Chemotherapy has played a minor role except for biotherapeutic agents used for metastatic disease.

Methods. From January 1989 to January 1996, 37 patients with renal cell carcinoma and inferior vena cava tumor thrombus underwent surgical resection. The 27 men and 10 women had a median age of 57 years (range, 29 to 78 years). Thirty-six patients presented with symptoms; 21 had hematuria. Distant metastases were present in 12 patients. Tumor thrombi extended to the infrahepatic inferior vena cava (n = 16), the intrahepatic inferior vena cava (n = 16), the suprahepatic inferior vena cava (n = 3), and into the right atrium (n = 2). All tumors were resected by inferior vena cava isolation and, when necessary, extended hepatic mobilization and Pringle maneuver, with primary or patch closure of the vena cavotomy. Cardiopulmonary bypass was necessary in only 2 patients with intraatrial thrombus.

Results. Complications occurred in 11 patients, and 1 patient died 2 days postoperatively of a myocardial infarction (mortality, 2.7%). Twenty patients are alive; overall 2- and 5-year survival rates were 61.7% and 33.6%, respectively. For patients without lymph node or distant metastases (stage IIIa), 2- and 5-year survival rates were 74% and 45%, respectively. The presence of distant metastatic disease (stage IV) at the time of operation did not have a significant adverse effect on survival, as reflected by 2- and 5-year survival rates of 62.5% and 31.3%, respectively. Lymph node metastases (stage IIIc) adversely affected survival as there were no long-term survivors.

Conclusions. Resection of an intracaval tumor thrombus arising from renal cell carcinoma can be performed safely and can result in prolonged survival even in the presence of metastatic disease. In our experience, extracorporeal circulatory support was required only when the tumor thrombus extended into the heart.     

Central opioid receptors differentially regulate the nalmefene-induced suppression of ethanol- and saccharin-reinforced behaviors in alcohol-preferring (P) rats.

The exact opioid-sensitive receptors participating in EtOH-seeking behaviors remains unclear. Previous studies have reported higher densities of micro-opioid receptor binding in the nucleus accumbens (NACC) of P relative to NP rats; however, no differences were seen in delta-receptor binding. In contrast to the NACC, substantially lower levels of micro-receptor binding have been observed in the ventral tegmental area (VTA) of both P and NP rats, albeit no line differences have been observed. In the present study, opioid receptors in the NACC, VTA, and hippocampus were evaluated for their capacity to regulate both EtOH- and saccharin-motivated behaviors in the genetically selected alcohol-preferring (P) rat. To accomplish this, nalmefene, an opiate antagonist with preferential binding affinity for the micro-opioid receptor was unilaterally or bilaterally infused during concurrent availability of 1 h daily EtOH (10% v/v) and saccharin (0.025 or 0.050% w/v) solutions. Rats performed under a two-lever fixed ratio (FR) schedule in which four responses on one lever produced the EtOH solution, and four on a second lever produced the saccharin solution. The results demonstrated that when responding maintained by both EtOH and saccharin are matched at basal levels, unilateral (1-60 microg) or bilateral (0.5-10 microg) microinjections of nalmefene into the NACC produced selective dose-dependent reductions on responding maintained by EtOH. Unilateral (40, 60 microg) and bilateral (10 microg) VTA infusions were also observed to selectively reduced EtOH responding; however, greater nalmefene doses were required and the magnitude of suppression on EtOH responding was markedly less compared with the NACC. The greater sensitivity of nalmefene to suppress EtOH responding in the NACC is likely due to the greater number of opioid receptors in the NACC relative to the VTA. Only bilateral infusion of the 40 microg dose in the NACC and VTA suppressed responding maintained by both EtOH and saccharin. In contrast, intrahippocampal infusions dose dependently suppressed EtOH- and saccharin-maintained responding over a range of doses (1-20 microg). The present study provides evidence that nalmefene suppresses EtOH-motivated behaviors via blockade of opioid receptors within the NACC and VTA, and under various dose conditions both reinforcer and neuroanatomical specificity can be observed.