Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Strategies in bioreactor performance studies

As bioreactor performance is the result of the interactions between physiology and physical transport phenomena an adequate strategy for design and analysis of bioreactors has to be based on this interaction network. The paper summarizes this systematic approach based on a holistic view called “integrating strategy”. The strategy is outlined for the case of baker's yeast processing, where especially the comparability of kinetic models including the concept of “dynamic flux” is critically discussed showing that macro- and microkinetics have similar potential in describing bioprocesses according to the formal macroapproach.     

Activation of human basophils through the IL-8 receptor.

IL-8 and its structural analogs derived from blood platelets have been proposed as stimuli of IgE-independent basophil activation. In order to clarify the mechanism of action of these peptides, we examined the effects of pure IL-8, connective tissue-activating peptide III (CTAP-III), neutrophil-activating peptide 2 (NAP-2), and platelet factor 4 (PF-4) on blood basophils with and without pretreatment by IL-3, which modulates mediator release. After pretreatment with IL-3, significant histamine release was observed with 10(-8) M and 10(-7) M IL-8 and 10(-7) M NAP-2, but not with the other peptides. At higher concentrations (10(-6) M), however, all IL-8 analogs, as well as the unrelated cationic peptides poly-D-lysine, histone VS, and lysozyme, induced histamine release to variable degrees. Binding and competition studies with [125I]IL-8 revealed specific IL-8R on basophils from a patient with chronic myelogenous leukemia and normal individuals. From 3500 to 9600 receptors with a mean Kd value of 0.15 nM were found on average per chronic myelogenous leukemia and normal basophil, respectively. NAP-2 weakly competed for IL-8 binding. IL-8 and, to a lesser extent, NAP-2 led to a transient rise of cytosolic free calcium concentration ([Ca2+]i), which was independent of a preexposure to IL-3. IL-8 prevented the [Ca2+]i rise induced by NAP-2, but did not influence [Ca2+]i responses to other agonists, e.g. C5a, C3a, or platelet-activating factor. IL-8 induced [Ca2+]i changes and histamine release in IL-3-primed basophils were pertussis toxin sensitive. CTAP-III or PF-4 did not compete for IL-8 binding, did not induce [Ca2+]i changes, and did not influence the [Ca2+]i response to IL-8 and NAP-2. This study shows that IL-8 and NAP-2 activate human basophils by a receptor-mediated mechanism similar to that operating in neutrophils. At high concentrations histamine release can also be induced by cationic peptides by a mechanism that does not involve the IL-8R, and probably depends on cationic interactions.     

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engineering approach to mixing quantification in bioreactors

Homogeneity-time is defined and introduced as the criterion for mixing quality in bioreactors. The criterion could replace the mixing time, in the case, when more than one measuring point (sensors) is included in the measuring system. Results based on the homogeneity-time and the temperature pulse method, achieved in stirred tank reactors under aerated conditions as well as in a jet-mixed tank, are presented.List of Symbols C _p,p kJ/kg K Heat capacity of the pulse medium - C _p,s kJ/kg K Heat capacity of the reactor-medium - F m³/s Flow rate of the pulse-input - i Inhomogeneity - I _N Inhomogeneity-number - M (t) °C Ideal response curve - m deNumber of combinations for certain number of sensors acc. to Table 1 - n Number of sensor - _p kg/m³ Density of the pulse medium - kg/m³ Density of the tank medium - s ₁ °C Mean absolute deviation of the sensor temperatures related on the ideal response curve s₂ s Mean absolute deviation of the homogeneity-times related on the time achieved with 6 sensors - t s Time - t (i) s Homogeneity-time - t _ps s Starting time of tracer injection - t _PE s End time of tracer injection - T _E °C Mean medium temperature at the end of experiment - T _k °C Temperature at k-th sensor position - T _p °C Pulse temperature - T _s °C Mean medium temperature before the tracer injection - V _s m³ Tank volume before pulse input     

Suppression of anti-hapten antibody responses by hapten-specific cytolytic T lymphocytes: role of I-A-associated antigen on target B lymphocytes

Cytolytic T lymphocytes (CTL) specific for 2,4,6-trinitrophenyl (TNP) determinants suppress the effector phase of a secondary anti-TNP antibody responses of murine syngeneic spleen cells in vitro. The cells mediating this suppression are hapten-specific, H-2-restricted, and possess properties typical of CTL. Moreover, the targets of the suppression appear to be antigen-primed B lymphocytes that are recognized by CTL via soluble antigen bound noncovalently to their Ig receptors. The effect of the CTL can be blocked by the addition of monoclonal antibodies directed against I-A molecules but not I-E or H-EK-encoded molecules on the target B cells, even in strain combinations in which the CTL-B cell interaction is restricted only by the H-2K and I regions of the MHC. This result suggests that B lymphocyte-bound antigen tends to associate preferentially with I-A rather than H-2K/D-encoded determinants, and that the suppressive effect of the CTL population is attributable to the minor subset that recognizes hapten-modified Ia antigens. These findings are also discussed in terms of the possible immunoregulatory function of Ia-restricted CTL.     

Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cyle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells' position within the G1 period. Data are presented that deal with three interrelated topics: (1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. (2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1.(3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Suspension culture of myocardial cells from newborn rats.

Myocardial cells from newborn rats were held in a spinner type culture for 2 days and then explanted into culture flasks. Three main cell types were observed: single multipolar cells of embryonic type, cell aggregates containing 10 to 50 connected cells, and bipolar cells retaining some adult characteristics. Except for the latter, up to 95% of intact cells settled and were beating 6 hours after explantation. The percentage of fibroblast-like cells was drastically reduced when compared with conventional cultures. Cell debris could be removed 2 hours after explantation by changing the culture medium, or more effectively by a density step centrifugation using Lymphoprep or Lymphoprep-Ficoll mixtures.