5‐HT2A Receptor Gene Promoter Polymorphism in Relation to Abdominal Obesity and Cortisol

Objective: There is considerable evidence that cortisol secretion is associated with obesity. The regulation of the 5‐hydroxytryptamine receptor 2A (5‐HT_2A) gene might play an essential role because it is involved in the control of cortisol secretion. Therefore, we examined the potential impact of the 5‐HT_2A ?1438G/A promoter polymorphism on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol, in 284 unrelated Swedish men born in 1944. Research Methods and Procedures: The subjects were genotyped by using polymerase chain reaction amplification of the promoter region of the gene for 5‐HT_2A followed by digestion of the reaction product with the restriction enzyme MspI. Results: The frequencies were 0.39 for allele ?1438A and 0.61 for allele ?1438G. Homozygotes for the ?1438G allele had, in comparison with ?1438A/A subjects, higher body mass index, waist‐to‐hip ratio, and abdominal sagittal diameter. Moreover, cortisol escape from 0.25‐mg dexamethasone suppression was found in subjects with the ?1438A/G genotype. Serum leptin, fasting insulin, and glucose, as well as serum lipids, were not different across the ?1438G/A genotype groups. Discussion: From these results, we suggest the possibility that an abnormal production rate of the 5‐HT_2A gene product might lead to the development of abdominal obesity. The pathophysiology could involve stress factors that destabilize the serotonin‐hypothalamic‐pituitary‐adrenal system in those with genetic vulnerability in the serotonin receptor gene.     

14C transfer between the spring ephemeral Erythronium americanum and sugar maple saplings via arbuscular mycorrhizal fungi in natural stands

We investigated in the field the carbon (C) transfer between sugar maple (Acer saccharum) saplings and the spring ephemeral Erythronium americanum via the mycelium of arbuscular mycorrhizal (AM) fungi. Sugar maple saplings and E. americanum plants were planted together in pots placed in the ground of a maple forest in 1999. Ectomycorrhizal yellow birches (Betula alleghaniensis) were added as control plants. In spring 2000, during leaf expansion of sugar maple saplings, the leaves of E. americanum were labelled with ¹⁴CO₂. Seven days after labelling, radioactivity was detected in leaves, stem and roots of sugar maples. Specific radioactivity in sugar maples was 13-fold higher than in yellow birches revealing the occurrence of a direct transfer of ¹⁴C between the AM plants. The quantity of ¹⁴C transferred to sugar maple saplings was negatively correlated with the percentage of ¹⁴C allocated to the storage organ of E. americanum. A second labelling was performed in autumn 2000 on sugar maple leaves during annual growth of E. americanum roots. Radioactivity was detected in 7 of 22 E. americanum root systems and absent in yellow birches. These results suggest that AM fungi connecting different understorey species can act as reciprocal C transfer bridges between plant species in relation with the phenology of the plants involved.     

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp. PCC6803 DnaB mini-intein.

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH(2)-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures approximately 14 degrees C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH(2) and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (Delta Delta G approximately 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.     

Distinct proteome pathology of circulating microparticles in systemic lupus erythematosus

Background

The pathogenesis of systemic lupus erythematosus (SLE) is poorly understood but has been linked to defective clearance of subcellular particulate material from the circulation. This study investigates the origin, formation, and specificity of circulating microparticles (MPs) in patients with SLE based on comprehensive MP proteome profiling using patients with systemic sclerosis (SSc) and healthy donors (HC) as controls.

Methods

We purified MPs from platelet-poor plasma using differential centrifugation of samples from SLE (n = 45), SSc (n = 38), and two sets of HC (n = 35, n = 25). MP proteins were identified and quantitated after trypsin digestion by liquid chromatography-tandem mass spectrometry. The abundance of specific proteins was compared between the groups using univariate statistics and false discovery rate correction for multiple comparisons. Specific proteins and protein ratios were explored for diagnostic and disease activity information using receiver-operating characteristic curves and by analysis of correlations of protein abundance with disease activity scores.

Results

We identify and quantitate more than 1000 MP proteins and show that a subpopulation of SLE-MPs (which we propose to call luposomes) are highly specific for SLE, i.e. not found in MP preparations from HC or patients with another autoimmune, systemic disease, SSc. In SLE-MPs platelet proteins and mitochondrial proteins are significantly diminished, cytoskeletal proteins deranged, and glycolytic enzymes and apoptotic proteins significantly increased.

Conclusions

Normal MPs are efficiently removed in SLE, but aberrant MPs, derived from non-lymphoid leukocytes, are less efficiently removed and abundantly produced leading to an altered MP proteome in SLE. The data suggest that an abnormal generation of MPs may partake in the pathology of SLE and that new diagnostic, monitoring, and treatment strategies targeting these processes may be advantageous.

     

New Cytotoxic Triterpenoid Saponins from the Roots of Albizia gummifera (J.F.Gmel.) C.A.Sm.

As part of our search for new bioactive saponins from Cameroonian medicinal plants, two new oleanane‐type saponins, named gummiferaosides D and E ( 1 and 2 ), along with one known saponin, julibroside J₈ ( 3 ), were isolated from the roots of Albizia gummifera. Their structures were established on the basis of extensive 1D‐ and 2D‐NMR (¹H‐ and ¹³C‐NMR, DEPT, COSY, TOCSY, NOESY, HSQC, HSQC‐TOCSY, and HMBC) and HR‐ESI‐MS studies, and by chemical evidence. The apoptotic effect of saponins 1  –  3 was evaluated on the A431 human epidermoid cancer cell. Flow cytometric analyses showed that saponins 1  –  3 induced apoptosis of human epidermoid cancer cell (A431) in a dose‐dependent manner.     

RANTES (CCL5) induces a CCR5-dependent accelerated shedding of syndecan-1 (CD138) and syndecan-4 from HeLa cells and forms complexes with the shed ectodomains of these proteoglycans as well as with those of CD44

We recently demonstrated that RANTES forms complexes with CCR5, syndecan-1 (SD-1), SD-4, and CD44 expressed by human primary macrophages and that SD-1 and SD-4 but neither CD44 nor SD-2 coimmunoprecipitate with CCR5. Here we show that RANTES directly binds in a glycosaminoglycan-dependent manner to SD-1, SD-4, and CD44. Moreover, RANTES accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells expressing CCR5 and, by contrast, has no effect on the constitutive shedding of CD44 from these cells. These accelerated sheddings are prevented by the MEK1/2 inhibitor, U0126, and by the protein kinase C inhibitor bisindolylmaleimide I. This indicates that both MAP kinase--and protein kinase C-dependent signaling pathways are involved in these RANTES-induced accelerated sheddings. RANTES also induces a decreased expression of SD-1 and SD-4 by HeLa cells expressing CCR5 and on the contrary an increased expression of CD44 by these cells. By contrast, RANTES neither accelerates the shedding of SD-1 and SD-4 ectodomains from HeLa cells lacking CCR5, nor changes the SD-1-, SD-4-, and CD44-plasma membrane expressions of these cells. CCR5 is therefore involved in the RANTES-induced accelerated shedding of SD-1 and SD-4 ectodomains. Nevertheless, the fact that RANTES stimulates in Hela cells (expressing or lacking CCR5) the mRNA synthesis of SD-1 and SD-4 indicates that the molecular events that follow the synthesis of these proteoglycans differ, according to the presence or not of CCR5. Finally, RANTES forms GAG-dependent complexes with the shed ectodomains of SD-1 and SD-4 as well as with those of CD44. The role of these events in the pathophysiology of RANTES deserves further study.     

Induction of hepatic drug-metabolising enzymes and tamoxifen metabolite profile in relation to administration route during low-dose treatment in nude rats

Tamoxifen is the most used anticancer drug and is approved for chemoprevention. Little is known about the enzyme inducing properties of low-dose regimens and the influence of route of administration. In this study, nude rats received 5 mg/kg/day of tamoxifen orally or a 50 mg continuous-release pellet subcutaneously. The mRNAs for cytochrome P450-enzymes (CYPs), flavin-containing monooxygenase 1 (FMO1) and phase II drug-metabolising enzymes were quantified by real-time RT-PCR. Tamoxifen and metabolite concentrations were measured using HPLC. We observed a significant increase in CYP3A18 and FMO1 mRNA expression levels in the orally treated animals, whereas the increase in CYP3A2 expression did not reach statistical significance (p = 0.057). No significant induction of enzyme expression was observed in rats that received subcutaneous (S.c.) treatment. After 33 days the serum levels of 4-hydroxytamoxifen (4OHtam), tamoxifen and N-desmethyltamoxifen (NDtam) in orally treated animals were 1.8 ± 0.7, 11.1 ± 3.2 and 11.4 ± 3.8 ng/ml, respectively. In subcutaneously treated animals, tamoxifen and N-desmethyltamoxifen were detected in tissues, but not in serum. These data demonstrate that in contrast to the subcutaneous administration, low-dose oral tamoxifen induced tamoxifen-metabolising enzymes. Furthermore, the different routes of administration resulted in different serum and tissue levels of tamoxifen and metabolites.     

Probing the link between citrate and malonyl-CoA in perfused rat hearts

Little is known about the sources of cytosolic acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of fatty acid oxidation in the heart. We tested the hypothesis that citrate provides acetyl-CoA for malonyl-CoA synthesis after its mitochondrial efflux and cleavage by cytosolic ATP-citrate lyase. We expanded on a previous study where we characterized citrate release from perfused rat hearts (Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846-E856, 2000). In the present study, we show that citrate release rates, ranging from 6 to 22 nmol/min, can support a net increase in malonyl-CoA concentrations induced by changes in substrate supply, at most 0.7 nmol/min. In experiments with [U-(13)C](lactate + pyruvate) and [1-(13)C]oleate, we show that the acetyl moiety of malonyl-CoA is derived from both pyruvate and long-chain fatty acids. This (13)C-labeling of malonyl-CoA occurred without any changes in its concentration. Hydroxycitrate, an inhibitor of ATP-citrate lyase, prevents increases in malonyl-CoA concentrations and decreases its labeling from [U-(13)C](lactate + pyruvate). Our data support at least a partial role of citrate in the transfer from the mitochondria to cytosol of acetyl units for malonyl-CoA synthesis. In addition, they provide a dynamic picture of malonyl-CoA metabolism: even when the malonyl-CoA concentration remains constant, there appears to be a constant need to supply acetyl-CoA from various carbon sources, both carbohydrates and lipids, for malonyl-CoA synthesis.