Purification and physical properties of hexokinase from human erythrocytes

A bulk purification is described for hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from human erythrocytes. Following a 110,000-fold purification from 40 litres of blood, 5 mg protein with a specific activity of 22 units/mg were obtained. On application of various separation techniques, the enzyme activity co-migrated with the main protein component. The physical properties, such as the relative molecular mass of 108,000 and sedimentation coefficient of 5.5 S, are similar to those of the enzyme from human heart. In particular, there is a correspondence in the conformational response to glucose 6-phosphate as shown by an association of the enzyme promoted by this metabolite.     

Evidence for the monomeric nature of thymosins

According to gel-filtration experiments, alpha- and beta-thymosins appear to form oligomers, which are 4-5-fold larger than the corresponding polypeptides. However, on analysis by sedimentation equilibrium ultracentrifugation, prothymosin alpha and thymosin beta 4 showed relative molecular masses of 12,800 and 4600, which are close to the values calculated from their amino acid sequences, confirming their existence in solution as discrete monomeric entities.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

Cytochrome c oxidase inhibition in the rice weevil Sitophilus oryzae (L.) by formate, the toxic metabolite of volatile alkyl formates

Volatile alkyl formates are potential replacements for the ozone-depleting fumigant, methyl bromide, as postharvest insecticides and here we have investigated their mode of insecticidal action. Firstly, a range of alkyl esters, ethanol and formic acid were tested in mortality bioassays with adults of the rice weevil, Sitophilus oryzae (L.) and the grain borer, Rhyzopertha dominica (F.) to determine whether the intact ester or one of its components was the toxic moiety. Volatile alkyl formates and formic acid caused similar levels of mortality (LC(50) 131-165 micromol l(-1)) to S. oryzae and were more potent than non-formate containing alkyl esters and ethanol (LC(50)>275 micromol l(-1)). The order of potency was the same in R. dominica. Ethyl formate was rapidly metabolised in vitro to formic acid when incubated with insect homogenates, presumably through the action of esterases. S. oryzae and R. dominica fumigated with a lethal dose of ethyl formate had eight and 17-fold higher concentrations of formic acid, respectively, in their bodies than untreated controls. When tested against isolated mitochondria from S. oryzae, alkyl esters, alcohols, acetate and propionate salts were not inhibitory towards cytochrome c oxidase (EC 1.9.3.1), but sodium cyanide and sodium formate were inhibitory with IC(50) values of 0.0015 mM and 59 mM, respectively. Volatile formate esters were more toxic than other alkyl esters, and this was found to be due, at least in part, to their hydrolysis to formic acid and its inhibition of cytochrome c oxidase.     

Chemoproteomics reveals Toll-like receptor fatty acylation

Background

Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.

Results

A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands.

Conclusions

This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. S-palmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.

     

Spatial and temporal aspects and the interplay of Grb14 and protein tyrosine phosphatase-1B on the insulin receptor phosphorylation

Background

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14?

Results

Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2^Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line.

Conclusions

Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling.

     

Comparsion of staining characteristics of Toto bodies

Toto bodies are eosinophilic structures that resemble the cells of the superficial cell layer of the oral epithelium. Toto bodies commonly are associated with inflammatory gingival and other mucosal lesions including pyogenic granuloma, irritational fibroma, epulis fissuratum, peripheral giant cell granuloma and inflammatory hyperplastic gingivitis. We evaluated staining characteristics of Toto bodies to establish their origin and to identify their significance in lesions. We investigated pyogenic granuloma, fibroma and leukoplakia with epithelium that exhibited Toto bodies after hematoxylin and eosin (staining. Sections were stained with Alcian blue, periodic acid-Schiff and Ayoub-Shklar stains to evaluate staining intensity and distribution. More Toto bodies were found in pyogenic granuloma than in fibroma and leukoplakia. PAS and Alcian blue staining exhibited mild intensity and did not establish the origin of Toto bodies. High staining intensity and diffuse distribution of stain was observed using Ayoub-Shklar staining, which indicated that Toto bodies originate from keratin.     

Cyclic gas exchange in the giant burrowing cockroach, Macropanesthia rhinoceros: effect of oxygen tension and temperature

The giant burrowing cockroach, Macropanesthia rhinoceros, is endemic to north-eastern Australia and excavates a permanent burrow up to 1m deep into soil. Using flow-through respirometry, we investigated gas exchange and water loss at three different oxygen tensions (21%, 10% and 2% at 20 degrees C) and temperatures (10, 20 and 30 degrees C at 21% oxygen). M. rhinoceros employ cyclic gas exchange (CGE) making the species by far the largest insect known to engage in discontinuous ventilation. CGE featured rhythmic bursts of CO(2) dispersed among inter-burst periods of reduced output. CGE was most commonly observed at 20 degrees C and degraded at <10% oxygen. Mild hypoxia (10% oxygen) resulted in a lengthening of the burst period by approximately two-fold; this result is complementary to oxygen consumption data that suggests that the burst period is important in oxygen uptake. When exposed to severe hypoxia (2% oxygen), CGE was degraded to a more erratic continuous pattern. Also, during severe hypoxia, total water loss increased significantly, although CO(2) release was maintained at the same level as in 21% oxygen. During CGE, an increase in temperature from 10 to 20 degrees C caused both water loss and CO(2) output to double; from 20 to 30 degrees C, CO(2) output again doubled but water loss increased by only 31%.