Molecular basis for hypertension in the "type II variant" of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess (AME) is a heritable form of hypertension in which cortisol acts as a potent mineralocorticoid. The type I variant results in a severe clinical and biochemical phenotype and arises because of mutations in the gene encoding the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2), an enzyme responsible for the peripheral inactivation of cortisol to cortisone. Only mild abnormalities of cortisol metabolism have been found in the type II variant of AME, suggesting that it may be a separate gene defect. In an extensive consanguineous Sardinian pedigree affected with "type II" AME, a novel homozygous point mutation (C945T) was found in the human 11beta-HSD2 gene in four affected individuals. Thirteen family members were heterozygous for the resultant R279C amino acid substitution. The LOD score of linkage of the mutation to the disease was 3.23. Expression of the 11beta-HSD2 mutant cDNA resulted in an enzyme with reduced maximum velocity, but similar substrate affinity, compared with activity of the wild-type cDNA. Affected individuals were >30 years of age and had both mineralocorticoid hypertension and evidence of impaired metabolism of cortisol to cortisone. The heterozygote state was phenotypically normal but was associated with subtle defects in cortisol metabolism. AME represents a spectrum of mineralocorticoid hypertension with severity reflecting the underlying genetic defect in the 11beta-HSD2 gene; classification into distinct subtypes is inappropriate. Hypertensive populations should be screened to identify the prevalence of milder defects in 11beta-HSD2 in patients currently labeled as having "essential" hypertension.     

Greenhouse and field evaluations of transgenic canola against diamondback moth, shape Plutella xylostella, and corn earworm, shape Helicoverpa zea

Canola (Brassica napus L.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis (Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea (Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and 90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and 95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995–1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non-transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.     

Regulation of tubulin polypeptides and microtubule function: Rki1p interacts with the β-tubulin binding protein Rbl2p

Genetic analysis of microtubule functions in the yeast Saccharomyces cerevisiae suggests that cells manage the levels and activities of the tubulin polypeptides. These reactions may be involved in protein folding, formation of the heterodimer, and maintenance of the appropriate balance between α- and β-tubulin. One protein involved in these functions is Rbl2p, which forms a complex with β-tubulin. Here we describe the identification of a novel yeast gene, RKI1, that interacts genetically with RBL2. Deletion of rki1 causes conditional defects in microtubule assembly and cell growth. Rki1p can be isolated in a complex containing Rbl2p. The results support the existence of cellular mechanisms for regulating microtubule function through the tubulin polypeptides. Received: 8 August 1998 / Accepted: 13 September 1998     

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ~130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.     

Photoinhibition in differently coloured juvenile leaves of Syzygium species

Photoinhibition, as measured by the dark-adapted chlorophyll a fluorescence ratio Fv/Fm, was assessed in Syzygium moorei, a species with dark green juvenile leaves, Syzygium corynanthum, which has light green juvenile leaves, and two species with pink-red juvenile leaves (Syzygium wilsonii and Syzygium luehmannii). All plants were glasshouse-grown (maximum PPFD 1500 mol m^-2 s^-1) under optimum nutrition and water.When measured at midday, dark-adapted Fv/Fm ratios of juvenile leaves gradually increased in all species as percentage of full leaf expansion (% FLE) increased. Fluorescence measurement 3h after sunset or pre-dawn also showed a developmental effect on Fv/Fm, with juvenile leaves of S. luehmannii and S. wilsonii showing much lower Fv/Fm at all stages of development. Dark-adapted Fv/Fm, values in both juvenile and mature leaves generally never exceeded 0.8 at any stage in any of the species.Courses of Fv/Fm on sunny days showed greater diurnal photoinhibition in green juvenile (c. 50% FLE) leaves of S. moorei (24%) and S. corynanthum (36%) than in mature leaves of the previous flush in these species (<10%). Diurnal photoinhibition was statistically similar (18-24%) in pink-red juvenile and green mature leaves of S. luehmannii and S. wilsonii. Re-positioning juvenile leaves of S. wilsonii horizontally increased diurnal photoinhibition.Exposure of leaves to a standard mild photoinhibitory light treatment (30 min at 1000 mol m^-2s^-1) showed that juvenile leaves of all species had a lower percentage of high energy state quenching (qE) and a higher percentage of photoinhibitory quenching (qI) than mature leaves.     

Short communication. Do anthocyanins play a role in UV protection of the red juvenile leaves of Syzygium?

The biological function of juvenile leaves pigmented with anthocyanin is poorly understood. The role anthocyanins play in UV protection was assessed in juvenile leaves of two Syzygium species (S. luehmannii and S. wilsonii) which contain high anthocyanin concentrations. HPLC was used to separate UV-absorbing anthocyanins from other soluble UV-absorbing phenolic compounds. The isolated anthocyanins (predominantly malvidin-3,5-diglucoside) contributed little to the total absorbance of UV-A and UV-8 radiation. This was because the non-acylated anthocyanins only effectively absorbed shortwave UV-B radiation and the strong absorbance by other compounds. These results suggest that the UV protection hypothesis is not valid for anthocyanins in juvenile Syzygium leaves.