Rationally designed non-peptides: variously substituted piperazine libraries for the discovery of bradykinin antagonists and other G-protein-coupled receptor ligands.

Molecular modeling studies of potent decapeptide bradykinin antagonists suggested the de novo design of peptide mimetics based on a 1,2,3,4-tetrasubstituted 1,4-piperazin-6-one scaffold. These de novo-designed antagonists exhibited only modest potency (IC50 approximately 55 microM) on a cloned human B2 receptor and antagonist activity in an in vitro human-cell functional assay. The success of these structures led to the creation of prototype libraries based on variously substituted 1,4-piperazine scaffolds, which allowed a rapid and general search of pharmacophores attached to a piperazine scaffold. The parent piperazinedione structures and fully reduced piperazine libraries differ from recently reported diketopiperazine libraries in the use of diverse nonnatural amino acids, on-resin-submonomer synthesis to provide more diverse N-substituted structures, and the adaptation of simultaneous ring closure and resin cleavage to drive the formation of highly hindered amide bonds. Using this chemistry, a rationally directed non-peptide library of approximately 2500 N,N'-disubstituted piperazines and piperazinediones was synthesized and screened for ligand affinity on bradykinin, neurokinin, and opioid receptors. A number of lead structures were identified. Notably, a bradykinin antagonist lead, CP-2458, with good receptor selectivity and antagonist activity in human-cell assays was identified and is undergoing optimization by traditional and combinatorial methods.

Evaluation of immunoglobulins from plant cells.

Expression of cDNA constructs encoding full-length mouse immunoglobulin chains with their native leader sequences or fusion constructs substituting the native leader with a pre-pro sequence derived from Saccharomyces cerevisiae yielded blocked N-termini on the gamma chain or the correct amino terminal sequence on the mature kappa chain. Lectin binding assays revealed that assembled immunoglobulin complexes contained a glycosylated heavy chain. The attached glycan was resistant to digestion by endoglycosidase H and its lectin binding pattern was distinguishable from that of the mammalian glycan. The results indicated processing of the immunoglobulin carbohydrate in the tobacco Golgi to yield a complex oligosaccharide. Secretion of antibody by protoplasts isolated from regenerated transgenic plants or from suspension callus cells was demonstrated by pulse-chase labeling experiments. When purified, the tobacco-produced antibody was found to possess the antigen binding and catalytic properties of the murine monoclonal antibody. Kinetic parameters (Km, Ki, Vmax, and kcat) of the tobacco-derived antibody were comparable to those of the mouse-derived antibody. The results in general show that the endomembrane system of tobacco cells possesses cognate mechanisms for the recognition of diverse leader sequences. These signals can be used to initiate the assembly, processing, and secretion by plant cells of complex foreign proteins.

A comparison of temperatures in canine solid tumours during local and whole-body hyperthermia administered alone and simultaneously.

Temperature measurements were made in canine solid tumours during whole-body hyperthermia (WBH) alone, local hyperthermia alone and local hyperthermia given simultaneously with WBH. During the plateau phase of WBH alone, mean intratumoral temperature ranged from 41.3 +/- 0.2 degrees C to 41.7 +/- 0.1 degrees C and was statistically lower (P = 0.0028) and more variable than rectal temperature, which ranged from 42.0 +/- 0.02 degrees C to 42.1 +/- 0.03 degrees C. The temperature distribution in solid tumours during WBH is more uniform than during local hyperthermia. The simultaneous administration of whole-body and local hyperthermia in five dogs resulted in increased tumour temperatures in comparison to WBH and in more uniformly increased tumour temperatures in comparison to local hyperthermia alone. Median intratumoral temperatures (+/- 95% confidence intervals) resulting from local hyperthermia alone and local hyperthermia given simultaneously with WBH were 39.9 degrees C (39.7-40.1) and 42.9 degrees C (42.6-43.1), respectively, and were statistically different (P = 0.0012). Local applied power requirements to meet predetermined intratumoral temperature limits were decreased by 50% (P = 0.011) in dogs undergoing combined local/whole-body hyperthermia versus local hyperthermia alone. Dogs tolerated the combination of local and WBH without complication.

Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells.

The gene encoding the complete glycoprotein of vesicular stomatitis virus (VSV, Indiana serotype G protein) with potential asparagine-linked glycans at amino acid residues 179 and 338 was inserted into a baculovirus transfer vector pAcYM1, derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promotor and expressed by the derived recombinant viruses to high levels in Spodoptera frugiperda cell lines. The principal product was the glycosylated version of the G protein, although some alternative (including probable degradation) forms of the protein were also observed. Similar recombinant viruses were prepared with deletion of one, the other, or both glycosylation sites of the VSV G protein. All forms expressed VSV G protein derivatives and mediated cell fusion and the production of syncytia at low pH. The fusogenic properties of the VSV G protein expressed on the surface of insect cells was prevented using anti-VSV sera, or by elevating the pH above 6.2. A reduction of the pH to 5.5, or 5.0, accelerated the rate of syncytia formation.

Whole body hyperthermia in dogs using a radiant heating device: effect of surface cooling on temperature uniformity.

Rectal and subcutaneous temperatures were measured during a total of 10 whole body hyperthermia treatments conducted in six dogs. During five of the treatments skin cooling, by means of initiating air flow through the radiant heating device, was necessary during the plateau phase because rectal temperature exceeded the target value. Skin cooling was not necessary in the other five treatments. Although the rectal temperatures were similar in all 10 treatments, extensive and rapid subcutaneous temperature non-uniformity, of approximately 4 degrees C, developed during treatments where skin cooling was necessary. During the treatments where skin cooling was not necessary, the subcutaneous temperature remained approximately equal to the rectal temperature. These data indicate that the environment in the radiant heating device during the plateau phase can have a profound effect on the temperature at superficial sites, which is not reflected by the temperature measured at deeper sites. The temperature at superficial sites should be measured during whole body hyperthermia to assure that the prescribed heat dose is administered to the largest percentage of body mass possible. Active skin cooling during whole body hyperthermia should be avoided if possible.

The combination of surgery and radiation in the treatment of cancer. A review.

Although radiation and surgery have been combined for the treatment of cancer in humans and animals since the 1920s, little has been written about the methods of combining radiation and surgery and the efficacy of this combination for the treatment of animal tumors. This article reviews the rationale for combining radiation and surgery for the treatment of cancer and the ways in which these two modalities can be combined with emphasis placed on the advantages and disadvantages of preoperative and postoperative radiotherapy. The role of preoperative and postoperative irradiation for the treatment of various animal tumors is discussed. Directions for future clinical trials are pointed out. Finally, the importance of surgeons and radiation oncologists communicating with each other and participating in cooperative treatment methods is stressed.

Evaluation of citrate-phosphate-dextrose-adenine as a storage medium for packed canine erythrocytes.

Packed canine red blood cells (RBCs) stored in the anticoagulant-preservative solution citrate-phosphate-dextrose-adenine (CPDA-1) were studied at 1, 10, 20, 30, and 40 days. The extracellular concentrations of potassium and sodium, erythrocyte mean corpuscular volume, and osmotic fragility increased during storage (P less than 0.05). There was a decrease in the pH, plasma concentration of glucose, and erythrocyte concentrations of 2,3-diphosphoglycerate (2,3-DPG) and adenosine-5'-triphosphate (P less than 0.05). Erythrocyte 2,3-DPG concentration decreased by 54% within the first 24 hours of storage (P less than 0.001). Posttransfusion viability (PTV) decreased from 90% on day 1 to 46% on day 40 (P less than 0.05). The PTV of the RBCs stored for 10 and 20 days complied with the Food and Drug Administration (FDA) standard. Although there are marked biochemical and hematologic changes in stored packed red blood cells (pRBCs), 20-day-old units may be expected to be of acceptable quality. The sharp decrease in 2,3-DPG concentration suggests a reduction in oxygen carrying capacity in erythrocytes stored as pRBCs. Hyperkalemia occurs during storage of pRBCs and does not appear to be associated with high intraerythrocytic potassium concentrations.

Use of insulation to reduce extremity temperature nonuniformity during whole body hyperthermia in dogs.

Previously we have shown in dogs that tibial bone marrow and s.c. tissue temperatures are lower than rectal temperature during the plateau phase of whole body hyperthermia with the use of a radiant heating device. In an attempt to increase thermal dose to these sites during whole body hyperthermia, we applied insulation to an extremity prior to the plateau phase of heating. We found that extremity insulation during whole body hyperthermia resulted in increased s.c. tissue and tibial bone marrow temperatures. With insulation, tibial bone marrow and rectal temperature were nearly equal but s.c. tissue temperature, although greater than without insulation, remained lower than rectal temperature. High efficiency extremity insulation or supplemental heating techniques may be necessary during whole body hyperthermia with the use of the radiant heat device in order to assure that extremities receive the prescribed thermal dose.

Positive serological evidence that Hantaan virus, the etiologic agent of hemorrhagic fever with renal syndrome, is endemic in Canada.

Hemorrhagic fever with renal syndrome is endemic in most parts of the world. The etiological agent in known as Hantaan virus. More than 2000 Canadian sera from all age groups and provinces were tested for the presence of antibody to this virus by the indirect immunofluorescent antibody technique. The frequency of seropositivity ranged from 0.3 to 4.0%, with the national average being 1.4%. Newfoundland and New Brunswick in the Atlantic region had the highest positivity, being higher than hyperendemic areas in Korea. The inland provinces of Ontario, Manitoba, and Alberta had only 0.5% positives to Hantaan virus. In contrast, approximately 1.5% of sera from Saskatchewan, Quebec, and British Columbia were positive. No correlation in positivity was found between geography or sex, but positivity did increase with age. The highest titres were found in the group aged 65 to 93 years.

Protease susceptibility of human A influenza virus polypeptides.

The proteolytic susceptibility of polypeptides of four antigenically distinct subtypes of influenza a virus strains of human origin was studied. The extent of degradation of polypeptide molecules of strains A/PR/8/34 (H0N1) (PR), A/FM/1/47 (H1N1), A/Singapore/1/57 (H2N2) and A/Hong Kong/8/68 (H3N2), assessed by densitometry of gels after sodium dodecylsulfate polyacrylamide gel electrophoresis was variable by treatment with trypsin. Also, sequential treatment of PR strain initially with phospholipase D followed by proteases of different specificities suggested differences in susceptibility of surface and internal polypeptide molecules. The significance of these results is discussed.

Hepatitis-associated antigen in sporadic cases of acute viral hepatitis.

Serum specimens collected during the period from September 1970 to April 1971 from hospitalized patients and apparently healthy staff members at risk were tested for the presence of hepatitis-associated antigen (HAA) by the parallel use of three techniques: the complement fixation test, crossover-electrophoresis and immuno-electronmicroscopy.Out of a total of 204 persons investigated 63 (30.9%) were found to harbour the antigen. The HAA-positive sera originated almost exclusively from sporadic cases of acute viral hepatitis (clinically diagnosed as "infectious hepatitis" or "serum hepatitis"). The great majority of the hepatitis cases studied had a history of drug addiction.