Insulin-regulatable phosphoproteins in 3T3-L1 adipocytes form detergent-insoluble complexes not associated with caveolin

Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, som: of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected P-32-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.

Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway

To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus or bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses. (C) 1998 Academic Press.

Matrix elements of U(2n) generators in a multishell spin-orbit basis. I. The del-operator MEs in a two-shell composite Gelfand-Paldus basis

This is the first in a series of three articles which aimed to derive the matrix elements of the U(2n) generators in a multishell spin-orbit basis. This is a basis appropriate to many-electron systems which have a natural partitioning of the orbital space and where also spin-dependent terms are included in the Hamiltonian. The method is based on a new spin-dependent unitary group approach to the many-electron correlation problem due to Gould and Paldus [M. D. Gould and J. Paldus, J. Chem. Phys. 92, 7394, (1990)]. In this approach, the matrix elements of the U(2n) generators in the U(n) x U(2)-adapted electronic Gelfand basis are determined by the matrix elements of a single Ll(n) adjoint tensor operator called the del-operator, denoted by Delta(j)(i) (1 less than or equal to i, j less than or equal to n). Delta or del is a polynomial of degree two in the U(n) matrix E = [E-j(i)]. The approach of Gould and Paldus is based on the transformation properties of the U(2n) generators as an adjoint tensor operator of U(n) x U(2) and application of the Wigner-Eckart theorem. Hence, to generalize this approach, we need to obtain formulas for the complete set of adjoint coupling coefficients for the two-shell composite Gelfand-Paldus basis. The nonzero shift coefficients are uniquely determined and may he evaluated by the methods of Gould et al. [see the above reference]. In this article, we define zero-shift adjoint coupling coefficients for the two-shell composite Gelfand-Paldus basis which are appropriate to the many-electron problem. By definition, these are proportional to the corresponding two-shell del-operator matrix elements, and it is shown that the Racah factorization lemma applies. Formulas for these coefficients are then obtained by application of the Racah factorization lemma. The zero-shift adjoint reduced Wigner coefficients required for this procedure are evaluated first. All these coefficients are needed later for the multishell case, which leads directly to the two-shell del-operator matrix elements. Finally, we discuss an application to charge and spin densities in a two-shell molecular system. (C) 1998 John Wiley & Sons.

Evidence for persistent dysfunction of wild-type aldosterone synthase gene in glucocorticoid-treated familial hyperaldosteronism type I

Background In familial hyperaldosteronism type I (FH-I), glucocorticoid treatment suppresses adrenocorticotrophic hormone-regulated hybrid gene expression and corrects hyperaldosteronism. Objective To determine whether the wild-type aldosterone synthase genes, thereby released from chronic suppression, are capable of functioning normally. Methods We compared mid-morning levels of plasma potassium, plasma aldosterone, plasma renin activity (PRA) and aldosterone : PRA ratios, measured with patients in an upright position, and responsiveness of aldosterone levels to infusion of angiotensin II (AII), for 11 patients with FH-I before and during long-term (0.8-14.3 years) treatment with 0.25-0.75 mg/day dexamethasone or 2.5-10 mg/day prednisolone. Results During glucocorticoid treatment, hypertension was corrected in all. Potassium levels, which had been low (< 3.5 mmol/l) in two patients before treatment, were normal in all during treatment (mean 4.0 +/- 0.1 mmol/l, range 3.5-4.6). Aldosterone levels during treatment [13.2 +/- 2.1 ng/100 ml (mean +/- SEM)] were lower than those before treatment (20.1 +/- 2.5 ng/100 ml, P < 0.05). PRA levels, which had been suppressed before treatment (0.5 +/- 0.2 ng/ml per h), were unsuppressed during treatment (5.1 +/- 1.5 ng/ml per h, P < 0.01) and elevated (> 4 ng/ml per h) in six patients. Aldosterone : PRA ratios, which had been elevated (> 30) before treatment (101.1 +/- 25.9), were much lower during treatment (4.1 +/- 1.0, P < 0.005) and below normal (< 5) in eight patients. Surprisingly, aldosterone level, which had not been responsive (< 50% rise) to infusion of AII for all 11 patients before treatment, remained unresponsive for 10 during treatment. Conclusions Apparently regardless of duration of glucocorticoid treatment in FH-I, aldosterone level remains poorly responsive to AII, with a higher than normal PRA and a low aldosterone : PRA ratio. This is consistent with there being a persistent defect in functioning of wild-type aldosterone synthase gene. (C) Rapid Science Publishers ISSN 0263-6352.

A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.

Copper(II) complexes of mono- and di-nucleating hexaamines

The potentially hexadentate polyamines N,N,N',N'-tetrakis(2-aminoethyl)ethane-1,2-diamine (L-1) and the octamethylated analogue N,N,N',N'-tetrakis(2-dimethylaminoethyl)ethane 1,2-diamine (L-2) have been complexed with copper(II) and the crystal structures of their complexes determined. A trigonal-bipyramidal co-ordination geometry for [Cu(HL1)][ClO4](3) was found where one aminoethyl arm is not co-ordinated. By contrast, a dinuclear structure of formula [(H2O)Cu(L-2)Cu(OH)](3+) was determined for the N-methylated analogue, where the hexaamine acts as a bridging ligand between the two square-pyramidal metal centres. Electronic and EPR spectroscopy are both consistent with these structures being maintained in solution.

New stereoselective routes to macrocyclic ligands

Reaction of bis(ethane-1,2-diamine)copper(II) with acetaldehyde and nitromethane in methanol leads, stereoselectively, to the new macrocyclic complex (trans-5(R),7(R),12(S),14(S))-tetramethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecane)copper(II) perchlorate alpha-[CuL1](ClO4)(2) in good yield. Reduction of the nitro groups affords the hexaamine (L-2), which was crystallized as [H4L2](ClO4)(4) . 2H(2)O and characterized by an X-ray crystal structure study (monoclinic P2(1)/n, a = 9.763(2) Angstrom, b = 12.1988(7) Angstrom, c = 13.036(2) Angstrom, beta = 105.668(7)degrees, Z = 2) and complexed with Cu-II to produce the complex beta-[Cu(H2L2)](ClO4)(4) . 2H(2)O, which has also been characterized by X-ray crystallography (monoclinic P2(1)/n, a = 9.717(4) Angstrom, b = 12.174(2) Angstrom, c = 13.036(5) Angstrom, beta = 106.51(2)degrees, Z = 2). Reaction of alpha-[CuL1](2+) with either basic hydrogen peroxide or dilute nitrous acid leads to mild reduction of the nitro groups to afford the ketoxime L-3 as its N-based isomeric Cu-II complexes, trans-I [CuL3](ClO4)(2) and trans-II [Cu(L-3)Cl]Cl . 7H(2)O, the latter of which has been characterized structurally: triclinic, <P(1)over bar> a = 10.8441(5) Angstrom, b = 11.6632(9) Angstrom, c = 11.8723(9) Angstrom, alpha = 113.634(7)degrees, beta = 95.744(5), gamma = 94.851(5)degrees Z = 2. Variations in the configurations of the coordinated amines in [CuL1](2+), [CuL2](2+), and [CuL3](2+) have a profound effect on the spectroscopy and electrochemistry of their complexes.

Young children, adolescents and alcohol - Part I: Exploring knowledge and awareness of alcohol and related issues

This article explores young children's and adolescents' views pertaining to: knowledge and awareness of alcohol and alcohol related issues; social situations in which. alcohol use is present; orientation to alcohol risk; perceived and actual alcohol use; social image and reputation; and short and long term health beliefs in relation to alcohol. Forty focus groups were conducted with 240 primary school students (118 males and 122 females) and 24 focus groups were conducted with 192 high school students (90 males and 102 females); the total being 64 focus groups comprising 432 school students. Participants ages ranged from five years three months to 16 years 10 months. The videotaped discussions revealed that approximately 75% of the primary school-aged children and almost all of the high school students reported that they had tasted alcohol. Parents were primarily responsible for providing the alcohol. Virtually all participants recognised and were able to correctly name a selection of alcoholic and non-alcoholic beverages, and levels of knowledge and awareness of the health and safety aspects of alcohol were relatively mixed. Presentation of bottles and cans was reported as being important in attracting young persons. These data suggest there is an urgent need for research addressed to the development of prevention/intervention education curriculum materials for use with primary school-aged children.

N-methylation of macrocyclic hexaaminecobalt(III) complexes

Reaction between formaldehyde and the pendant arm macrocyclic complex (trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine)cobalt(III) [CoL1](3+) yielded the diimine derivative trans-6,13-dimethyl-6.13-bis(methyleneamino)-1,4,8,11-tetraazacyclotetradecane (L-3) as its cobalt(III) complex. Reduction of the imines has been achieved with NaBH4 and the meso and rac cobalt(III) complexes of trans-6,13-dimethyl-6,13-bis(methylamino)-1,4,8,11-tetraazacyclotetradecane (L-5) have been prepared. Crystal structures of the macrocyclic complexes [CoL1][ClO4](3), [CoL3][ClO4](3) and meso-[CoL5][ClO4](3).2H(2)O were determined and some unusual structural, spectroscopic and electrochemical variations observed going from the parent hexaamine [CoL1](3+) to [CoL3](3+) (diimine) and ultimately to [CoL5](3+) (bis-N-methylated hexaamine).

Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules

Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands, As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.

Thermochromism and structure of piperazinium tetrachlorocuprate(II) complexes

The tetrachlorocuprate(II) ion can crystallize in two different structures with the piperazinium dication (pipzH(2)). Both structures contain discrete CuCl42- species. A yellow compound (pipzH(2))[CuCl4]. 2H(2)O (1) is monoclinic (C2/c, Z = 4, a = 10.538(3) Angstrom, b = 7.4312(5) Angstrom, c = 17.281(4) Angstrom, beta = 111.900(10)degrees) and contains the CuCl42- ion as a distorted tetrahedron. A green compound (pipzH(2))(2)[CuCl4]. Cl-2. 3H(2)O (2) is triclinic (P (1) over bar, Z = 2, a = 9.264(3) Angstrom, b = 10.447(2) Angstrom, c = 11.366(2) Angstrom, alpha = 68.38 degrees, beta = 82.86(2)degrees, gamma = 83.05(2)degrees) and contains the CuCl42- ion with a square planar geometry. This latter compound shows thermo/photochromism, changing from green to yellow upon heating or laser irradiation.

Trunculins G-I: New norsesterterpene cyclic peroxides from a southern Australian marine sponge, Latrunculia sp.

A Latrunculia sp, collected off Port Phillip Bay, Victoria, returned three new norsesterterpene cyclic peroxides. Trunculins G (9), H (10) and I (11) were isolated as their methyl esters (12), (13) and (14) respectively. Gross structures for these new trunculins were assigned on the basis of spectroscopic analysis, while the absolute stereochemistry about the cyclic peroxide terminus was established by application of the Horeau and Mosher procedures.