Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Pathogenicity of Fusarium oxysporum Isolates from Malaysia and F. oxysporuin f. sp. elaeidis from Africa to Seedlings of Oil Palms (Elaeis guineensis)

Pathogenicity tests with Fusarium oxysporum isolated form Malaysian oil palm were made with oil palms seedlings raised form Malaysian seed as well s with wilt-susceptible seedlings gown from African seed. Oil palm seedlings grown form Malaysian seed were also inoculated with African isolates of F. oxysporum f. sp. elaeidis and F. oxysporum var. redolens. The experiments were made under normal soil moisture conditions and under water stress. F. oxysporum f. sp. elaeidis isolates form Africa were pathogenic to oil palm seedlings from Malaysian seeds but the Malaysian F oxysporum isolates were non-pathogenic to plams grown from Malaysian seed or the wilt-susceptible palms from African seed. Seedlings from Malaysian seed proved to be highly susceptible to the vascular wilt disease caused by F. oxysporum f. sp. elaeidis as 75–90% of the palms were infected. The susceptibility of the palms from Malaysian seed varied with different African isolates tested. The Yaligimba isolate from Zaire which was found to be F. oxysporum var. redolens was the most virulent. Disease was more severe when oil palm seedlings were subjected to a period of water stress. The incidence of death in the seedlings under stress conditions was 45% as compared with only 15% for palms grown under normal conditions.     

Laryngeal constriction in normal humans during experimentally induced bronchoconstriction

Changes in the size of the glottis with bronchoconstriction were assessed in six normal subjects following inhalation of histamine or methacholine. Measurements were made during both tidal breathing and panting at 2-3 Hz. The midexpiratory size of the glottis was decreased by a mean of 8% during bronchoconstriction compared with control during tidal breathing. Changes in midinspiratory size were inconsistent. During panting, the glottic size was unchanged from inspiration to expiration but decreased in 7 of 15 studies during bronchoconstriction. The decreases in expiratory size of the glottis during quiet breathing would lead to an elevated laryngeal resistance coupled with an increased lower airway resistance. Although this seems to be a paradoxical laryngeal response, it may contribute to maintaining hyperinflation during bronchoconstriction, thereby effectively enlarging the lower airways.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Modification of glutathione levels in C3H/10T1/2 cells and its relationship to benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide-induced cytotoxicity

Levels of reduced glutathione (GSH) in C3H/10T1/2 cells were selectively altered to determine what quantitative role GSH transferase-catalyzed conjugation plays in regulating the cytotoxic effects of benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene, anti-diol epoxide). A 65% decrease in 10T1/2 cell GSH content from 0.16 mM (control cell GSH concentration) to 0.06 mM was accompanied by a 46% decrease in the anti-diol epoxide LD80; a 98% increase in GSH content resulted in a 44% increase in anti-diol epoxide LD80. This nonlinear relationship between changes in cellular GSH concentration and anti-diol epoxide LD80 was directly relatable to the nonlinear change in the rate of anti-diol epoxide conjugation which was catalyzed by 10T1/2 cell GSH transferases. Purified 10T1/2 cell cytosol catalyzed the GSH conjugation of anti-diol epoxide to yield a GSH conjugation product with a distinct UV absorbance spectrum; the apparent GSH Km for this cell cytosol-catalyzed reaction was 0.20 mM. Variations in the cellular GSH concentration around the GSH Km resulted in a nonlinear change in the amount of anti-diol epoxide-GSH conjugate formed, and a reciprocal change in the amount of free anti-diol epoxide available for cytotoxic alkylation events. These results clarify in quantitative, biochemical terms how GSH transferase-catalyzed conjugation can regulate the level of an electrophilic carcinogen metabolite in a biological system.     

Effects of superovulatory doses of pregnant mare serum gonadotropin on oocyte quality and ovulatory and steroid hormone responses in rats

Immature rats (aged 28 days) were injected with 4, 20, or 40 IU pregnant mare serum gonadotropin (PMSG) and sacrificed every 6 or 12 hr. Control rats (4 IU) ovulated between 60 and 72 hr, whereas rats given superovulatory doses of PMSG (20 and 40 IU) ovulated between 24 and 72 hr. The oocyte count from the superovulated rats increased slightly between 24 and 36 hr and markedly between 48 and 72 hr. Degenerated oocytes were recovered 48 and 36 hr after administration of 20 and 40 IU PMSG, respectively. Thereafter, the proportion of degenerated oocytes was dose dependent and reached a maximum at 72 (30.9%, 20 IU) and 60 hr (61.0%, 40 IU). 17β-estradiol content of the superovulated ovaries increased significantly (P < 0.01) from 36 hr and was maximal at 60 (20 IU) or 54 hr (40 IU), when compared to the control regimen. Administration of 40 IU PMSG resulted in a biphasic increase of progesterone content with the peaks at 36 and 60 hr. Androgen content of the superovulated ovaries was lower than control levels during the first 36 hr but was significantly (P < 0.01) higher thereafter. The results suggest that these alterations in the steroid response (particularly androgens) from 36 hr onward following superovulation may be responsible for the coincidental occurrence of abnormal oocytes, possibly by disturbing the specific intrafollicular steroid environment essential for complete maturation. In addition, oocyte aging that is due to earlier activation by the exogenous luteinizing hormone activity may be a contributing factor.     

A simple purification of calmodulin by reversed phase chromatography with hydrophobic polymer 3520

A new adsorption chromatography procedure for the purification of calmodulin from bovine brain was developed using polymeric adsorbent 3520. Calmodulin was first isolated by DEAE-Cellulose column chromatography and further purified to apparent homogeneity following elution with 50% ethanol from the adsorbent column. Polyacrylamide gel electrophoresis showed one band either in the presence of Ca2+ or EGTA. The polymeric adsorbent 3520 is a non-polar polymer lacking exchangeable groups. The selective adsorption of calmodulin is based on hydrophobic interaction within the matrix, and is Ca2+ independent. Neither high salt (0.5 M NaC1) nor EGTA (5 mM) was able to elute the CaM from the adsorption column whereas ethanol (50%) eluted it completely. This method is simple to use and it provides highly purified calmodulin with high yield.