Characterization of serological properties of polyclonal antibodies produced against enzymes involved in the L-selective cleavage of hydantoin derivatives

Summary Polyclonal antibodies were produced against the highly purified enzymes L-hydantoinase, hydantoin-racemase and L-N-carbamoylamino acid amidohydrolase of Arthrobacter aurescens DSM 3747. In order to exploit these antibodies for basic research (molecular biology) or bioengineering (process development), the serological properties had to be characterized. Both, the hydantoinase- and carbamoylase-antibodies were observed to be monofunctional, whereas the hydantoin-racemase-antibody was found to be additionally specific against the L-hydantoinase. Monospecificity was realized after affinity chromatography. Investigations on serological crossreactions with several linear- and cyclic amidases (e.g. hydantoinases) as well as hydantoin-racemases are demonstrated in this paper.Deticated to Prof. Dr. Klaus Mosbach on the occation of his 60th birthday.     

Monoclonal antibodies and idiotypic network activation for ovarian carcinoma

Antibodies can be processed by the B- and T-cell systems and may lead to a selective activation of the immune system. The network structure of the immune system implicates the possibility of a selective immunization by the activation of idiotypic cascades. In a retrospective analysis, patients with advanced ovarian carcinoma, who had received MAb, against the cancer-associated antigen CA125 for diagnostic purposes, were analyzed for the production of anti-idiotypic antibodies, survival rate, and immunological effects. Furthermore, we started a prospective and randomized study for ovarian cancer patients, using a different antigen, TAG72, for the induction of idiotypic cascades. Our first results on 58 patients with advanced ovarian carcinomas showed that the induction of anti-idiotypic-antibodies against OC125 mimicking the TAA Class III CA125 leads to a prolongation of the survival rate, and, in extended stages, to an induction of antitumoral immunity, and that the induction of idiotypic cascades is also possible for different antigens like TAG72. Summarizing the activation of idio-typic network cascades seems to be a very effective way of intervention in the immune system of patients with advanced stages of ovarian carcinoma. A prospective study of the adjuvant approach seems to be necessary.     

The control of respiration in wheat (Triticum aestivum L.) leaves

The aim of this work was to discover whether the respiration of wheat (Triticum aestivum L. cv. Huntsman) leaves, transferred to darkness after 7 h photosynthesis, showed an initial period of wasteful respiration. For young and old leaves, CO₂ production and O₂ uptake after 7 h photosynthesis were up to 56% higher than at the end of an 8-h night. The maximum catalytic activities of citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) at the end of the day did not differ from those at the end of the night. Changes in the contents of glucose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and -ketoglutarate did not as a group parallel the changes in the rate of respiration. The detailed distribution of label from [U-¹⁴C] sucrose supplied to leaves in the dark was similar at the end of the day and the end of the night. No correlation was observed between the rates of leaf respiration and extension growth. It is argued that the higher rate of respiration at the beginning of the night cannot be attributed to wasteful respiration.Abbreviation RQ respiratory quotient We thank Dr H. Thomas and Professor C.J. Pollock, Institute for Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, UK for their generous help in measuring leaf extension. R.H.A. thanks the Science and Engineering Research Council for a studentship.     

Localization of dopamined D1-receptors in vertebrate retinae

We used the fluorescent labelled dopamine D₁-receptor antagonist Bodipy-SCH 23390 for the cellular localization of D₁-ligand binding sites in the retinae of different vertebrates (teleosts, Xenopus, turtle, rat and rabbit). Competition experiments with unfixed cryosections of fish retina were performed to characterize the binding conditions of Bodipy-labelled SCH 23390. Tissue bound [³H]SCH 23390 was displaceable with increased amounts of bodipy-SCH 23390. The pharmacological specificity of the D₁ fluorescent antagonist was determined by competition experiments with an excess of unlabelled SCH 23390. This treatment significantly reduced the level of fluorescence of the retina confirming the specificity of the binding. We observed a homogeneously distributed fluorescence signal in both plexiform layers in unfixed cryosections of fish, frog, turtle, rat and rabbit. Similar staining intensities of both plexiform layers were found in frog, turtle, rat and rabbit retina. In teleosts, the label of the outer plexiform layer was markedly more intense. Non-specific label was associated with photoreceptor outer and inner segments. The specific labelling of both plexiform layers indicates a mismatch of dopamine releasing and D₁-binding sites, and suggests a possible extrasynaptic localization of the D₁-receptor. The physiological significance of the observed distribution of D₁-ligand binding sites is discussed with respect to the role of dopamine in controlling adaptational processes in the retina.     

Computer determination of perfusion patterns in pulmonary capillary networks

Hanger, Christopher C., Robert G. Presson, Jr., Osamu Okada,Steven J. Janke, John J. Watkins, Wiltz W. Wagner, Jr., and Ronald L. Capen. Computer determination of perfusion patterns in pulmonarycapillary networks. J. Appl. Physiol.82(4): 1283-1289, 1997.Individual pulmonary capillaries are notsteadily perfused. By using in vivo microscopy, it can readily bedemonstrated that perfusion continually switches between capillarysegments and between portions of the network within a single alveolarwall. These changes in capillary perfusion occur even when upstream pressure and flow are constant. Flow switching between capillary segments in the absence of hemodynamic changes in large upstream vessels suggests that capillary perfusion patterns could be random. Tocalculate the probability that perfusion patterns could occur bychance, it is necessary to know the total number of possible perfusionpatterns in a given capillary network. We developed a computer programthat can determine every possible perfusion pattern for any givencapillary network, and from that information we can calculate whetherperfusion of individual segments in the network is random. With theresults of the computer program, we have obtained statistical evidencethat some capillary segments in a network are nonrandomly perfused.

     

Capillary recruitment and transit time in the rat lung

Presson, Robert G., Jr., Thomas M. Todoran, Bracken J. DeWitt, Ivan F. McMurtry, and Wiltz W. Wagner, Jr.Capillary recruitment and transit time in the rat lung.J. Appl. Physiol. 83(2): 543-549, 1997.Increasing pulmonary blood flow and the associated rise incapillary perfusion pressure cause capillary recruitment. The resultingincrease in capillary volume limits the decrease in capillary transittime. We hypothesize that small species with relatively high restingmetabolic rates are more likely to utilize a larger fraction ofgas-exchange reserve at rest. Without reserve, we anticipate thatcapillary transit time will decrease rapidly as pulmonary blood flowrises. To test this hypothesis, we measured capillary recruitment andtransit time in isolated rat lungs. As flow increased, transit timedecreased, and capillaries were recruited. The decrease in transit timewas limited by an increase in the homogeneity of the transit time distribution and an increased capillary volume due, in part, to recruitment. The recruitable capillaries, however, were nearly completely perfused at flow rates and pressures that were less thanbasal for the intact animal. This suggests that a limited reserve ofrecruitable capillaries in the lungs of species with high restingmetabolic rates may contribute to their inability to raiseO₂ consumption manyfold abovebasal values.

     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.