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.

The human temporal cortex: Characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles

Immunocytochemical techniques were used to examine the distribution of neurons immunoreactive (-ir) for nitric oxide synthase (nNOS), somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), calbindin (CB) and calretinin (CH), in the inferotemporal gyros (Brodmann's area 21) of the human neocortex. Neurons that colocalized either nNOS or SOM with PV, CB or CR were also identified by double-labeling techniques. Furthermore, glutamate receptor subunit profiles (GluR1, GluR2/3, GluR2/4, GluR5/6/7 and NMDAR1) were also determined for these cells. The number and distribution of cells containing nNOS, SOM, NPY, PV, CB or CR differed for each antigen. In addition, distinct subpopulations of neurons displayed different degrees of colocalization of these antigens depending on which antigens were compared. Moreover, cells that contained nNOS, SOM, NPY, PV, GB or CR expressed different receptor subunit profiles. These results show that specific subpopulations of neurochemically identified nonpyramidal cells may be activated via different receptor subtypes. As these different subpopulations of cells project to specific regions of pyramidal calls, facilitation of subsets of these cells via different receptor subunits may activate different inhibitory circuits. Thus, various distinct, but overlapping, inhibitory circuits may act in concert in the modulation of normal cortical function, plasticity and disease.

Nitric oxide, a potent vasodilator of the aortic anastomosis in the estuarine crocodile, Crocodylus porosus

The effects of five neuropeptides (CGRP, SOM, SP, NPY, VIP), L-NAME (nitric oxide synthase inhibitor), and adrenaline on the contractile tone of the aortic anastomosis in the estuarine crocodile, Crocodylus porosus, were investigated. None of the neuropeptides, which had previously been found to be present in the aortic anastomosis, had any direct effect on the tension developed by ring preparations. L-NAME itself significantly increased the basal tone of the vascular ring preparations, suggesting a tonic release of nitric oxide in the preparation. Adrenaline produced concentration-dependent vasoconstrictions that were counteracted by profound reflex vasodilatations that were susceptible to blockade by L-NAME. Immunohistochemistry revealed the presence of nitric oxide synthase and tyrosine hydroxylase-containing (indicating the presence of a adrenergic innervation) nerve fibres in the adventitia and adventitio-medial border of the aortic anastomosis. These data demonstrate opposing actions of adrenaline and nitric oxide on the vascular smooth muscle in the anastomosis of the C. porosus. The morphology of the anastomosis, with the extremely thick muscular vessel wall, suggests a sphincter-like function for this vessel that could be controlled mainly by adrenergic and nitrergic mechanisms, (C) 2001 Academic Press.

Hydrofluoric acid pre-treatment for improving 13C CPMAS NMR spectral quality of forest soils in south-east Queensland, Australia

Hydrofluoric acid (HF) was used to pre-treat forest soils of south-east Queensland for assessing the effectiveness of iron (Fe) removal, carbon (C) composition using C-13 cross-polarisation (CP) with magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) before and after the HF pre-treatment, and the improvement of C-13 CPMAS NMR spectra. Soil samples were collected from 4 experimental sites of different soil types, harvest residue management or prescribed burning, and tree species. More than 86% of Fe was in all soil types removed by the HF treatment. The C-13 NMR spectral quality was improved with increased resolution, especially in the alkyl C and O-alkyl C regions, and reduced NMR run-time (1-5 h per sample compared with >20 h per sample without the pre-treatment). The C composition appeared to alter slightly after the pre-treatment, but this might be largely due to improved spectrometer conditions and increased resolution leading to more accurate NMR spectral integration. Organic C recovery after HF pre-treatment varied with soil types and forest management, and soluble soil organic matter (SOM) could be lost during the pre-treatment. The Fourier Transform-Infrared (FT-IR) spectra of HF extracts indicated the preferential removal of carboxylic C groups during the pre-treatment, but this could also be due to adsorbed water on the mineral matter. The NMR spectra revealed some changes in C composition and quality due to residue management and decomposition. Overall, the HF treatment was a useful pre-treatment for obtaining semi-quantitative C-13 CPMAS NMR spectra of subtropical Australian forest soils.

Nitrate in soil humic acids revealed by N-14 nuclear magnetic resonance spectroscopy

Ecosystem management such as plant residue retention and prescribed burning can significantly affect soil organic matter (SOM) composition and, thereby, the closely associated carbon (C) and nitrogen (N) cycling processes, which underpin terrestrial ecosystem productivity and sustainability. Humic acid (HA) is an important SOM component and its chemical composition has attracted much attention. Here we report the first application of N-14 nuclear magnetic resonance (NMR) spectroscopy to soil HA study, revealing the surprising existence of nitrate-N and ammonia-N in the HAs. This newly discovered HA nitrate-N, though in a relatively low concentrations, is closely related to soil N availability and responsive to plant residue management regimes in contrasting forest ecosystems. The HA nitrate-N may be a useful and sensitive biochemical indicator of SOM quality in response to different ecosystem management regimes.

Composition and quality of harvest residues and soil organic matter under windrow residue management in young hoop pine plantations as revealed by solid-state C-13 NMR spectroscopy

Solid-state C-13 nuclear magnetic resonance (NMR) with cross-polarisation (CP) and magic-angle-spinning (MAS) was used to: (a) examine the changes in carbon (C) composition of windrowed harvest residues during the first 3 years of hoop pine plantations in subtropical Australia; (b) assess the impacts of windrowed harvest residues on soil organic matter (SOM) composition and quality in the 0-10 cm soil layer. Harvest residues were collected from 0-, 1-, 2- and 3-year-old windrows of ca. 2.5 m width (15 m apart for 0-, 1- and 2-year-old sites and 10 m apart for 3-year-old site). Soils from the 0 to 10 cm soil layer were collected from the 1-, 2- and 3-year-old sites. The 13C NMR spectra of the harvest residues indicated the presence of lignin in the hoop pine wood, foliage and newly incorporated organic matter (NIOM). Condensed tannin structures were found in the decay-resistant bark, small wood and foliage, but were absent in other residue components and SOM. The NMR spectra of small wood samples contained condensed tannin structures because the outer layer of bark was not removed. NIOM showed a shift from foliage-like structures (celluloses) to lignin-type structures, indicating an incorporation of woody residues from the decomposing harvest residues. Suberins were also present in the small wood, foliage and bark. The 13C CP NMR spectra of SOM indicated that in areas where windrows were present, SOM did not show compositional changes. However, an increase in SOM quality under the windrows in the second year after their formation as characterised by the alkyl C/O-alkyl C (A/O-A) ratio was mainly due to inputs from the decomposition of the labile, readily available components of the windrowed harvest residues. (C) 2002 Published by Elsevier Science B.V.

How does residue management impact soil organic matter composition and quality under Eucalyptus globulus plantations in southwestern Australia?

This study investigated the influence of harvest residue management practices on soil organic matter (SOM) composition and quality from two second-rotation Eucalyptus globulus plantations in southwestern Australia, using solid-state 13C nuclear magnetic resonance (NMR) spectroscopy with cross-polarisation and magic-angle-spinning (CPMAS). Soil samples (0–5 cm) were collected every 12 months for 5 years from two sites that had contrasting soil types and fertility. Harvest residue management treatments established at both sites were (a) no harvest residues; and (b) double harvest residues. The use of 13C CPMAS and DD NMR spectroscopy enabled the successful non-destructive detection of SOM quality changes in the two E. globulus plantations. Relative intensities of 13C CPMAS NMR spectral regions were similar at both sites, and for both harvest residue treatments, indicating that SOM composition was also similar. Dipolar dephasing (DD) NMR spectra revealed resonances in SOM assigned to lignin and tannin structures, with larger resonances in the carbonyl and alkyl C regions that were indicative of cuticular material, enabling detection of changes in SOM quality. Retention of double harvest residues on the soil surface increased the soil quality compared with removal of all harvest residues at both sites as indicated by the NMR aromaticities, but this was most noticeable at Manjimup, which had greater initial soil fertility.