Triterpene glycosides from Schefflera octophylla.

In addition to 3-epi-betulinic acid, three triterpene glycosides were isolated from leaves of Schefflera octophylla. The structures of the glycosides have been determined as 28-O-[alpha-L-rhamnopyranosyl(1----4)-O-beta-D-glucopyranosyl(1----6)-be ta-D- glucopyranosides of 3 alpha-hydroxy-lup-20(29)-ene-23,28-dioic acid, 3 alpha,11 alpha- dihydroxy-lup20(29)-ene-23,28-dioic acid and 3-epi-betulinic acid by spectroscopic data and chemical transformations. The last two compounds were found for the first time in the plant kingdom.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.     

Enzyme-linked immunosorbent assays for detecting antibodies to Shiga-like toxin I,Shiga-like toxin II,andEscherichia coli O157:H7 lipopolysaccharide in human serum

Shiga-like toxin-producingEscherichia coli O157:H7 are important causes of bloody diarrhea and hemolytic uremic syndrome. To facilitate the epidemiologic study of these organisms, we developed enzyme-linked immunosorbent assays (ELISAs) for antibodies to Shiga-like toxin I (SLT I), Shiga-like toxin II (SLT II), andE. coli O157 lipopolysaccharide (LPS). We tested serum samples from 83 patients in two outbreaks ofE. coli O157:H7 diarrhea and from 66 well persons. Forty-three patients (52%) had at least one serum sample positive for anti-O157 LPS antibodies; among 26 culture-confirmed patients, 24 (92%) had at least one positive serum sample. Two (3%) of 66 control sera had positive anti-O157 LPS titers. ELISA results for SLT I and II were compared with those of HeLa cell cytotoxicity neutralization assays on both patient and control sera. Neutralization assays detected anti-SLT I antibodies in at least one serum sample from each of 17 (20%) patients and 7 (10.6%) controls, while 16 (19%) patients and 7 controls had positive titers by anti-SLT I ELISA. Although all serum samples, including control sera, showed nonspecific neutralization of SLT II, no antibody titers to SLT II were detected by either neutralization or ELISA. These results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays. Both the ELISAs and neutralization assays are insensitive in detecting infected patients. However, the ELISA for antibodies toE. coli O157 LPS is both sensitive and specific, and may be more useful than assays for antitoxic antibodies in detecting persons withE. coli O157:H7 infection.     

Ananain: a novel cysteine proteinase found in pineapple stem

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Identification of host range determinants in the Rhizobium species MPIK3030

Summary R. meliloti primarily nodulates Medicago sativa but cannot nodulate Macroptilium atropurpureum. By introducing an 11.4 kb region into R. meliloti from the Symplasmid of Rhizobium strain MPIK3030, the host range of the R. meliloti transconjugants were shown to be extended to M. atropurpureum, one of the hosts of MPIK3030 but not normally nodulated by R. meliloti. The region responsible for host range extension was isolated by mass conjugating a clone bank from MPIK3030 into the R. meliloti wild type, and subsequent screening for nodulation on M. atropurpureum. Using deleted derivatives of a plasmid reisolated from endosymbiotic bacteria, the host range region was further narrowed down to three EcoRI fragments. Tn5 mutagenesis allowed the isolation of three discrete regions on an 11.4 kb section, which are involved in the extension of host range to M. atropurpureum. Finally, complementation experiments performed with R. meliloti common nod and hsn mutants indicated that none of the genes involved in the early steps of nodulation, including host-range functions, can be complemented by genes carried on the 11.4 kb fragment derived from MPIK3030.     

Evidence for a pool of non-recycling transferrin receptors in peripheral sheep reticulocytes

Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.