[18O]-Oxygen incorporation reveals novel pathways in spiroacetal biosynthesis by Bactrocera cacuminata and B. cucumis

The origins of the oxygen atoms in 1,7-dioxaspiro[5.5]undecane (1) and hydroxyspiroacetal (2) from Bactrocera cacuminata, and in 2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (3) and hydroxyspiroacetal (4) from B. cucumis, have been investigated by incorporation studies from both [18O2]-dioxygen and [18O]-water. Combined GC-MS examination and high-field NMR analysis have demonstrated that all oxygen atoms in 1 and 2 from B. cacuminata are dioxygen derived, but in contrast, the spiroacetals 3 and 4 from B. cucumis incorporate one ring oxygen from water and one ring oxygen (and the hydroxyl oxygen in 4) from [18O2]-dioxygen. These results reveal not only the generality of monoxygenase mediation of spiroacetal formation in Bactrocera sp., but also an unexpected complexity in their biosynthesis. A general paradigm accommodating these and other observations is presented.

2,6,,9-trioxabicyclo[3.3.1]nona-3,7-dienes and 2,4,6,8-tetraoxaadamantanes: Novel chiral spacer units in macrocyclic polyethers

The unusual chiral heterocyclic systems, trioxabicyclo[3.3.1]nona-3,7-dienes (bridged bisdioxines), are incorporated as novel spacer molecules into macrocyclic polyether ring systems of various sizes (8, 9 as well as 11-15) by cyclocondensation reaction of the! bisacid chloride 4b or bisesters 6,7 and 10, with several ethylene glycols. The 2:2 macrocycles 12-14 are obtained in approximately 50:50 mixtures of diastereomers. These conclusions are mainly based on HPLC data presented in Table I as well as X-ray analyses of (1R,5R)-8c (space group Pbca, a = 10.163(3) Angstrom, b = 18.999(4) Angstrom, c = 36.187(10) Angstrom, V = 6987(3) Angstrom(3), Z = 8, d(calc) = 1.218 g cm(-3), 6974 reflections, R = 0.0553.), mesolrac-11 (space group P (1) over bar, a = 10.472(5) Angstrom, b = 16.390(5) Angstrom, c = 17.211(5) Angstrom, alpha = 98.69(2)degrees, beta = 93.04(2)degrees, gamma = 98.52(2)degrees, V = 2879.3(18) Angstrom(3), Z = 2, d(calc) = 1.173 g cm(-3), 11,162 reflections, R = 0.0945) and meso-12 (space group P2(1)/c, a = 9.927(2), b = 18.166(3), c = 17.820(3) Angstrom, beta = 96.590(10)degrees, V = 3192.3(10)Angstrom(3), Z = 4, D-c = 1.109 g cm(-3), 3490 reflections, R = 0.0646). The 1:1 macrocycles 8b,c are also formed by intramolecular transesterification of the open-chain bisesters 7b,c and their formation is favored by the use of metal ions as templates. The bridged bisdioxine moieties in 8b and 12 are converted into the corresponding chiral tetra-oxaadamantane spacers to afford macrocycles 16 and 17. Preliminary metal ion complexation studies with selected species (8c, 11-14) were also performed.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Kinematics of oblique collision and ramping inferred from microstructures and strain in middle crustal rocks, central Southern Alps, New Zealand

The hanging wall of the Alpine Fault near Franz Josef Glacier has been exhumed during the past similar to2-3 m.y. providing a sample of the ductilely deformed middle crust of a modem obliquely convergent orogen. Presently exposed rocks of the Pacific Plate are inferred to have undergone several phases of ductile deformation as they moved westward above a mid-crustal detachment. Initially they were transpressed across the outboard part of the orogen, resulting in oblate fabrics with a down-dip stretch. Later, they encountered the Alpine Fault, experiencing an oblique-slip backshearing on vertical planes. This escalator-like deformation tilted and thinned the incoming crust onto that crustal-scale oblique ramp. This style of hanging wall deformation may affect only the most rapidly uplifting, central part of the Southern Alps because of the low flexural rigidity of the crust in that region and its displacement over a relatively sharp ramp-angle at depth. A 3D transpressive flow affected mylonites locally near the fault, but their shear direction remained parallel to plate motion, ruling out ductile 'extrusion' as an important process in this orogen. Outside the mylonite zone, late Cenozoic shortening is inferred to be modest (30-40%), as measured from deformation of younger biotite grains. Oblique collision is dominated by translation on the Alpine Fault, and rocks migrate rapidly through the deforming zone, preventing the accumulation of large finite strains. Transpression may play a minor role in oblique collision. (C) 2001 Elsevier Science Ltd. All rights reserved.

Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope

Infection with group A streptococci (GAS) can lead to rheumatic fever (RF) and rheumatic heart disease (RHD) which are a major health concern particularly in indigenous populations worldwide, and especially in Australian Aboriginals. A primary route of GAS infection is via the upper respiratory tract, and therefore, a major goal of research is the development of a mucosal-based GAS vaccine, The majority of the research to date has focused on the GAS M protein since immunity to GAS is mediated by M protein type-specific opsonic antibodies. There are two major impediments to the development of a vaccine-the variability in M proteins and the potential for the induction of an autoimmune response. To develop a safe and broad-based vaccine, we have therefore focused on the GAS M protein conserved C-region, and have identified peptides, J8 and the closely related J8 peptide (J14), which may be important in protective immunity to GAS infection. Using a mucosal animal model system, our data have shown a high degree of throat GAS colonisation in B10.BR mice 24 h following intranasal immunisation with the mucosal adjuvant, cholera toxin B subunit (CTB), and/or diptheria toxoid (dT) carrier, or PBS alone, and challenge with the M1 GAS strain. However, GAS colonisation of the throat was significantly reduced following intranasal immunisation of mice with the vaccine candidate J8 conjugated to dT or J14-dT when administered with CTB. Moreover, J8-dT/CTB and J14-dT/CTB-immunised mice had a significantly higher survival when compared to CTB and PBS-immunised control mice. These data indicate that immunity to GAS infection can be evoked by intranasal immunisation with a GAS M protein C-region peptide vaccine that contains a protective B cell epitope and lacks a T cell autoepitope. (C) 2002 Published by Elsevier Science Ltd.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

Serum inhibin A and B concentrations during the menstrual cycle in mothers of spontaneous dizygotic twins

Dizygotic twinning in humans is influenced by genetic factors suggesting inherited variation affects follicle development and predisposes to double ovulations. In a previous study, we conducted a detailed examination of follicle development and variation in hormone concentrations during the menstrual cycle in mothers of DZ twins (MODZT) compared with an age-matched control group of mothers of singletons. We did not detect differences in FSH concentrations between mothers of twins and mothers of singletons. Serum inhibin concentrations were measured by a radioimmunoassay that did not distinguish between dimeric inhibin A and B forms and free inhibin alpha subunit. We therefore analyzed the samples from this study with specific assays to determine whether concentrations of inhibin A and B were different between MODZT and controls and therefore contribute to the twinning phenotype. There were no significant differences between MONT with single ovulations and control women in inhibin A and B concentrations during the cycle, including the critical period for the selection of the dominant follicle. These data suggest that the genetic cause of twinning is not associated with changes in FSH concentrations or recognised feedback mechanisms regulating FSH release.

Effect of solute lipophilicity on penetration through canine skin

Objective To investigate the effect of lipophilicity on the percutaneous penetration of a homologous series of alcohols through canine skin Design Skin harvested from Greyhound thorax was placed in Franz-type diffusion cells and the in vitro passage of radio-labelled (C-14) alcohols (ethanol, butanol, hexanol and octanol (Log P 0.19 - 3.0)) through separate skin sections was measured in replicates of five. Permeability coefficient (k(P), cm/h), maximum flux (J(max), mol/cm(2)/h) and residue remaining within the skin were determined. Results The k(P) increased with increasing lipophilicity (6.2 x 10(-4) +/- 1.6 x 10(-4) cm/h for ethanol to 1.8 x 10(-2) 3.6 x 10(-3) cm/h for octanol). Alcohol residues remaining within each skin sample followed a similar pattern. An exponential decrease in Jmax with increasing lipophilicity was observed. Conclusion Changes in canine skin permeability occur with increasing alcohol lipophilicity. This finding has practical consequences for the design of topical formulations and optimisation of drug delivery through animal skin.