Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes

Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutaraldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methemoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused an R----T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyanomethemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb4(O2)3, Hb4(O2)2 and Hb4(O2), as well as mixtures of these intermediates, could be monitored.     

Sex Differences in the Steepness of Dominance Hierarchies in Captive Bonobo Groups

Bonobos have a reputation as a female-dominated and egalitarian species. We examined the 2 aspects of dominance in 6 captive bonobo groups. Females do not consistently evoke submission from all males in all contexts. Though females occupy the highest-ranking positions in the dominance hierarchy, there are in each group males that obtain rather high ranks and are able to dominate ≥1 female. Thus female dominance is not complete and hierarchies can be better described as nonexclusive female dominance. We studied egalitarianism by measuring linearity and steepness of dominance hierarchies. The hierarchies of all groups are highly linear. Hierarchies among males are steeper than among females. On average, male bonobos are more despotic than females, but females too can have despotic relations, both with other females and with males. Hence one can call bonobos in captivity semidespotic rather than egalitarian.     

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.     

The functioning of cytochrome b in the electron transport to fumarate in Propionibacterium freudenreichii and Propionibacterium pentosaceum

Fumarase-free electron particles from Propionibacterium freudenreichii and P. pentosaceum were prepared by discontinuous sucrose gradient centrifugation, and the influence of 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO) and ultraviolet irradiation on the reduction of menaquinone and cytochrome b with l-lactate or glycerol-3-phosphate and the reoxidation by fumarate was studied. In the presence of HQNO the steady state reduction level of menaquinone during fumarate reduction was increased whereas the steady state reduction level of cytochrome b was decreased as compared with the reduction levels measured in the absence of HQNO. The steady state reduction level of menaquinone during electron transport to fumarate was not influenced by ultraviolet irradiation and the steady state reduction level of cytochrome b was decreased at increasing irradiation times. The data indicate that cytochrome b is involved in the electron transport to fumarate.Abbreviations HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide Visiting Professor at the Biological Laboratory     

Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.)

Summary An amylose-free potato mutant was isolated after screening 12,000 minitubers. These minitubers had been induced on stem segments of adventitious shoots, which had been regenerated on leaf explants of a monoploid potato clone after Röntgen-irradiation. The mutant character is also expressed in subterranean tubers and in microspores. Starch granules from the mutant showed a strongly reduced activity of the granule bound starch synthase and loss of the major 60 kd protein from the starch granules.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Complexity of T cell receptor recognition sites for defined alloantigens

Three monoclonal antibodies react with the T cell receptor on the tumor line HPB-ALL and in addition with 3 to 13% of human peripheral blood T cells of normal donors. These antibodies are shown to react with an epitope encoded by the V beta 5 family of T cell receptor beta-chain variable region gene segments. Cells expressing V beta 5 gene segments can have cytotoxic or helper function, be of the T4+ or T8+ phenotype, and have specificity for either class I or class II major histocompatibility complex alloantigens. Seven T cell clones were generated, which express V beta 5 and are specific for the HLA-A2 molecule. With the use of these clones, we illustrate how isoelectric focusing can be used to analyze T cell receptor alpha- and beta-chain structure. The seven clones recognize five distinct conformational determinants on HLA-A2. They procure different binding sites by the use of different alpha-chains, J beta sequences, or both.     

Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.