The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Studies on the factors influencing larval settlement in Balanus amphitrite and Mytilus galloprovincialis

Understanding of factors influencing settlement(attachment and metamorphosis) of marine invertebratelarvae is of great importance in aquaculture andcontrol of biofouling. The influence of two factors onsettlement of larvae was assessed from two separateinvestigations: 1, the influence of age (endogenousfactor) on cyprids of the barnacle Balanusamphitrite; and 2, the influence of a microbial film(exogenous factor) on pediveligers of the mussel Mytilus galloprovincialis.The settlement response of cypris larvae of B.amphitrite was found to be age-dependent. Oldercyprids responded more readily to settlement factorsthan newly molted ones. In M.galloprovincialis, competent pediveligers settled inresponse to a microbial filmed surface but not toan unfilmed surface. Moreover, a factor with MW of lessthan 5000 dalton, derived from culture medium of abacterial strain C1.1 (Pseudomonas-Alteromonasgroup), induced the settlement of M. galloprovincialis larvae.Thus, marine invertebrate larvae may require a periodof competence acquisition, during which they arepoorly responsive to settlement inducers. Uponacquisition of competence, larvae readily respondto external cues (e.g. microbial film, bacterialextracellular products).     

Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.     

Purification,primary structure,and evolution of cytochrome c-550 from the magnetic bacterium,Magnetospirillum magnetotacticum

Cytochrome c-550 was purified from Magnetospirillum magnetotacticum to an electrophoretically homogeneous state, and some of its properties were determined. The cytochrome showed absorption peaks at 528 and 409 nm in the oxidized form, and at 550, 521, and 414 nm in the reduced form. Its midpoint redox potential at pH 7.0 was determined to be +289 mV. The primary structure of cytochrome c-550 was determined. Cytochrome c is composed of 97 amino acid residues, and its molecular weight was calculated to be 10,873, including heme c. Its primary structure is very similar to those of Rhodospirillum fulvum and Rhodospirillum molischianum cytochromes c ₂, suggesting that M. magnetotacticum is phylogenetically related to photosynthetic bacteria.     

Oligo-glycine synthesis in an aqueous solution of glycine under oxidative conditions

Di-and tri-glycine were synthesized in 1M aqueous solution of glycine by bubbling for 90 hr with oxygen discharged in the path from an oxygen cylinder. The peptides were also produced by an incubation at 37°C of 2M glycine solution prepared with 75% hydrogen peroxide, and the yields were traced for 200 days. The final yields were about 0.25% and 0.01% for di-and tri-glycine, respectively. The solution at 166 days of incubation was applied to a Sephadex G 10 column, and the fractions around the top of the chromatogram were found to increase the intensity of ninhydrin color about 45 times after hydrolysis, indicating an existence of oligo-glycine. The solutions of 1M glycine and 0.5M diglycine prepared with 30% hydrogen peroxide were incubated at 37°C for 38 days, and di-and tetra-glycine were detected in the yields of 0.12% and 0.33%, respectively.     

Hypothalamic and hormonal control of photoperiodically induced vernal functions in the white-crowned sparrow,Zonotrichia leucophrys gambelii

Summary The effects of implantation of testosterone propionate (TP) in various sites in the hypothalamus on the photoperiodically induced vernal premigratory functions in the White-crowned Sparrows were investigated in order to assess the role of the hypothalamo-hypophysial-testicular axis in the induction of these responses.Implantation of glass capillary tubes containing TP in the basal infundibular nucleus (IN), in the median eminence, or in the pars distalis inhibited the photoperiodically induced increase in plasma levels of luteinizing hormone (LH), as measured by radioimmunoassay, and testicular growth. The effective implants significantly lowered the levels of LH in birds held on nonstimulatory short days. These TP implants apparently inhibited release from the pars distalis of both LH and follicle-stimulating hormone (FSH). It is concluded that the site of sensitivity in the negative feedback by testosterone is either the basal IN or the pars distalis, or both. The implants of TP that inhibited the increase in plasma LH and testicular growth completely did not prevent the birds from fattening.These investigations were supported, in part, by research grants from the National Institutes of Health (HD-6527) and National Science Foundation (BMS 79-13933) to Professor Donald S. Farner. This paper is based on a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the University of WashingtonThe author is grateful to Professor Donald S. Farner for his guidance throughout the course of these investigations. The assistance of Mr. Philip W. Mattocks, Jr. in performing radioimmunoassays is sincerely appreciated     

Immunological Studies of Betaine Aldehyde Dehydrogenase in Barley

The changes in the level of the protein for betaine aldehydedehydrogenase, which catalyzes the last step in the synthesisof glycinebetaine, were analyzed with antiserum raised againstSDS-denatured betaine aldehyde dehydrogenase from spinach. Inbarley leaves, the levels of betaine aldehyde dehydrogenaseprotein were found to be enhanced by the addition of 200 mMNaCl to the growth medium. These changes in the level of theenzyme protein corresponded to those in the activity of theenzyme, as described in our previous study (Arakawa et al. 1990).The extent of this enhancement was reduced when barley plantswere relieved from salt stress. An increase in the level ofthe protein was also induced by water stress, such as the withholdingof water or the addition of polyethylene glycol 6000. Betainealdehyde dehydrogenase protein was detected in etiolated leavesand roots, as well as in green leaves. In etiolated leaves,the level of betaine aldehyde dehydrogenase protein was notaffected by salt stress. ¹ This work was supported by a grant from the Bio-Media Projectof the Japanese Ministry of Agriculture, Forestry and Fisheries(BMP92-III-l-1).     

Neurochemical Studies on the Cerebellar Hypoplasia of Gunn Rat (Hereditary Hyperbilirubinemic Rat)

The cerebellar hypoplasia induced by hereditary hyperbilirubinemia in the Gunn rat was analyzed neurochemically and immunohistochemically. The antiserum against myelin basic protein was used to visualize the arborization of the fibers in the cerebellum. Arborization was very scarce in the affected lobes of the homozygous (jj) cerebellum. Na,K-ATPase activity did not show significant differences between the jj and the control (Jj) cerebellum. The concentration of norepinephrine in the jj cerebellum was about 1.5 times that of the control. However, the activation ratio of the Na,K-ATPase by norepinephrine and other catecholamines such as dopamine and isoproterenol was about twice as high as the basal activity, and no significant difference was observed between the jj and the Jj cerebella. The glutamic acid decarboxylase activity of the jj cerebellum did not differ significantly from that of the control.     

Amphotericin B resistance is recessive in Chinese hamster hybrid cells

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B-resistant (AMB^R) mutants were each hybridized to their parental amphotericin B-sensitive (AMB^S) V79 cells. All the hybrids derived from AMB^R/AMB^S fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMB^S/AMB^S hybrids. The AMB^R/AMB^S hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMB^S or AMB^S/AMB^S. The analysis of hybrids formed between mutant and wild-type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMB^S/AMB^R hybrids. Hybrids of AMB^R and AMB^R cells were all resistant, so that the three AMB^R mutants all fell into a single complementation group.