Apigenin 4′-O-β-d-glucoside 7-O-β-d)- glucuronide: The copigment in the blue pigment of Centaurea cyanus

The copigment present in the crystalline blue pigment isolated from Blue Boy cornflowers (Centaurea cyanus L.) was identified as apigenin 4′-O-β-glucoside 7-O-β-d-glucuronide. The NMR spectra of aryl glucuronides are discussed.     

Repetitive excitation of bursts of action potentials in the rat hippocampus following single shock stimulation

1. Post-stimulus time (PST) histograms of rat hippocampal cells were recorded in vivo following single-shock stimulation of the fornix. 2. The PST histograms displayed a series of peaks of decreasing amplitude, similar to damped oscillatory responses previously recorded in cats and rabbits. 3. The effect of increased background activity was investigated by recording histograms with concurrent pulse train stimulation of the contralateral hippocampus. The histograms showed a decreased latency to the onset of the second peak. 4. Damped oscillatory activity seen in the in vivo rat preparation could not be elicited in the in vitro rat slice preparation. Thus species differences cannot account for the absence in slice studies of this type of damped oscillatory activity. 5. We conclude that the level of spontaneous activity is one factor contributing to the genesis of multiple peaks in histograms in the in vivo preparation.     

Catecholamine-induced changes in transmembrane potential and temperature in normal and dystrophic hamster brown adipose tissue

Previous studies have shown that norepinephrine (NE) elicits trans-membrane potential changes in skeletal muscle cells from normal and dystrophic (BIO 14.6) hamsters, with the magnitude of these changes being significantly less in dystrophic cells. To determine if the decreased response of the dystrophic muscle cells reflects a more generalized phenomenon, the present study was designed to evaluate the effects of NE on membrane properties of brown adipocytes. $\text{In vivo}$ techniques using glass microelectrodes were similar to those used in the muscle studies. NE injection (2 to 5 μg/kg body wt, i.v.) into anesthetized hamsters was followed by membrane depolarization, the magnitude of which did not significantly differ in the dystrophic and normal adipocytes. For example, upon administration of 5 μg NE/kg body wt, the average depolarization was $\text{14.5 ± 1.3}\text{mV}\text{(}\text{X}\text{±}\text{S.E.}\text{)}$ for 20 dystrophic cells and 14.1 ± 1.8 mV for 18 normal cells. The depolarizations following i.v. infusion of isoproterenol and phenylephrine also had similar amplitudes in both normal and dystrophic cells. Despite this lack of difference in plasma membrane responses, NE induced a significantly smaller rise in interscapular brown fat temperature in the dystrophic (0.09°C) than in the normal hamsters (0.26°C) following administration of 5 μg NE/kg body wt. Thus, the decreased responsiveness to NE of dystrophic sarcolemma did not occur with the plasma membrane of brown adipocytes, although brown fat temperature changes in the dystrophic hamsters were decreased in amplitude.     

The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis

The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.     

The differential functional stability of various forms of bovine liver rhodanese

Bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was prepared in dilute solutions and subjected to conditions that led to a time-dependent loss of enzyme activity. The rate of this activity loss was found to be dependent upon the sulfur substitution state of the enzyme, and the presence or absence of the substrates, thiosulfate and cyanide. In the absence of excess substrates, free enzyme (E), and the covalent intermediate form of the enzyme bearing a divalent sulfur atom in the active site (ES), are of approximately equal functional stability. In comparison, E, in the presence of excess cyanide, was markedly more labile, while ES, supported by 10-50 mM thiosulfate, showed no significant loss of activity under any of the conditions tested. All the enzyme solutions were shown to be losing assayable protein from solution. However, it was demonstrated that, for rhodanese in the E form, the amount of protein lost was insufficient to account for the activity lost, and a marked decline in specific activity was observed. Enzyme in the ES form, whether supported by additional thiosulfate or not, did not decline in the specific activity, though comparable protein loss did occur from these solutions. Intrinsic fluorescence measurements of rhodanese in the ES form, before and after removal of the persulfide sulfur through the addition of cyanide, indicated that loss of enzymic activity was not accompanied by loss of the bound sulfur atom. Therefore, the stabilizing effect observed with thiosulfate could not be explained simply by its ability to maintain enzyme in the sulfur-substituted state. Since the concentration of thiosulfate employed in these experiments was insufficient to maintain all the enzyme in ES.S2O3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.     

Studies of the secondary structures of amelogenin from bovine tooth enamel

Circular dichroism and Fourier transform infrared spectroscopic studies of the major amelogenin protein of developing bovine tooth enamel in solution and in the solid state suggest a unique secondary structure containing beta-sheet and repetitive beta-turn structures. The repetitive beta-turn structure at the C-terminal end results from the unique primary structure of amelogenin.     

Immunosuppression in viral oncogenesis. III. Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness

Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus. We studied the effects of MV infection and MV-associated tumor-induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2. Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E. coli endotoxin. Neither live virus nor TISF alters the production or the responsiveness to IL 1. However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient. Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4. TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2. Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants. Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2. Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed. We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally. Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2.