Protein phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1_G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1_G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1_G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF- (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1_G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1_G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1_G subunit site-specific mutations will help clarify the exact role and regulation of PP-1_G site-specific phosphorylations on PP-1 catalytic function.     

Human MD-1 homologue is a BCG-regulated gene product in monocytes: its identification by differential display

BCG-CWS is a therapeutically potent immune activator which improves the prognosis of cancer patients. However, the targeting effector cells and molecules for BCG-CWS in the human immune system have not been determined. Here, we found that BCG-CWS activates human monocytes and concomitantly down-regulates expression of a human homologue of chicken MD-1 in the activated monocytes by differential display. According to a previous study, MD-1 forms a complex with the Toll family protein RP-105 on murine B cell lines to facilitate its stable expression. Thus, MD-1 may participate in regulation of innate immune activation on human monocytes. Our results, taken together with these recent findings regarding Toll family proteins, suggest that BCG-CWS acts on monocytes to modulate the human innate immune system via regulation of Toll family proteins.     

Proteomic analysis of human low‐density lipoprotein reveals the presence of prenylcysteine lyase,a hydrogen peroxide‐generating enzyme

The molecular mechanisms underlying the relationship between low‐density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid‐phase IEF has been used to resolve LDL proteins into well‐defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL‐associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol‐binding protein, and paraoxonase‐1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time‐dependent generation of H₂O₂ which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H₂O₂ generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.     

Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin''s high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method''s sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.     

Tetraketones: a new class of tyrosinase inhibitors

Twenty-eight tetraketones (1-28) with variable substituents at C-7 were synthesized and evaluated as tyrosinase inhibitors. Remarkably compounds 25 (IC(50)=2.06 microM), 11 (IC(50)=2.09 microM), 15 (IC(50)=2.61 microM), and 27 (IC(50)=3.19 microM) were found to be the most active compounds of the series, even better than both standards kojic acid (IC(50)=16.67 microM) and L-mimosine (IC(50)=3.68 microM). This study may lead to the discovery of therapeutically potent agents against clinically very important dermatological disorders including hyperpigmentation as well as skin melanoma.     

In vivo expression of the heat stable (estA) and heat labile (eltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.     

Zinc supplementation in children with cholera in Bangladesh: randomised controlled trial

Objective To investigate the impact of zinc supplementation in children with cholera.Design Double blind, randomised, placebo controlled trial.Setting Dhaka Hospital, Bangladesh.Participants 179 children aged 3-14 years with watery diarrhoea and stool dark field examination positive for Vibrio cholerae and confirmed by stool culture.Intervention Children were randomised to receive 30 mg elemental zinc per day (n=90) or placebo (n=89) until recovery. All children received erythromycin suspension orally in a dose of 12.5 mg/kg every six hours for three days.Main outcome measures Duration of diarrhoea and stool output.Results 82 children in each group completed the study. More patients in the zinc group than in the control group recovered by two days (49% v 32%, P=0.032) and by three days (81% v 68%, P=0.03). Zinc supplemented patients had 12% shorter duration of diarrhoea than control patients (64.1 v 72.8 h, P=0.028) and 11% less stool output (1.6 v 1.8 kg/day, P=0.039).Conclusion Zinc supplementation significantly reduced the duration of diarrhoea and stool output in children with cholera. Children with cholera should be supplemented with zinc to reduce its duration and severity.Trial registration Clinical trials {"type":"clinical-trial","attrs":{"text":"NCT00226616","term_id":"NCT00226616"}}NCT00226616.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.