The 18 kDa cytosolic acid phosphatase from bovine liver has phosphotyrosine phosphatase activity on the autophosphorylated epidermal growth factor receptor

In this paper we demonstrate that the cytosofic low-M_r acid phosphatase purified from bovine liver has phosphotyrosine protein phosphatase acitivity on ³²P-autophosphorylated epidermal growth factor (EGF) receptor. This activity was significantly inhibited by orthovanadate and p-hydroxymercuribenzoate; the latter result indicates that free sulfhydryl groups are required for phosphotyrosine phosphatase activity. The enzyme was active in a broad pH range, with maximum activity between pH 5.5 and 7.5. The apparent K_m for ³²P-EGF receptor dephosphorylation was 4 nM. The enzyme appeared to be specific for phosphotyrosine in that it dephosphorylated the autophosphorylated EGF receptor and L-phosphotyrosine, but not ³²P-Ser-casein, L-phosphoserine or L-phosphothreonine. These data suggest that the cytosolic low-M_r acid phosphatase might play a regulatory role in EGF receptor-dependent transmembrane signalling.     

Ionization and binding equilibria of papaverine in ionic micelles studied by 1H NMR and optical absorption spectroscopy

The binding of the vasodilator drug papaverine (PAV) to micelles of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS), cationic cetyltrimethylammonium chloride (CTAC) and anionic sodium dodecylsulfate (SDS) in aqueous solution was studied by 1H NMR and electronic absorption spectroscopy. In the presence of HPS or CTAC, the apparent pK(a) of PAV decreased by about 2 units, while it increased by about 2 units upon binding to SDS. However, the chemical shift patterns of both protonated (PAVH+) and deprotonated (PAV0) forms of PAV are not sensitive to the type of surfactant. The association constants were estimated as 5 +/- 2 M(-1) for PAVH+-CTAC, 8 +/- 3 M(-1) for PAVH+-HPS, (7 +/- 2) x 10(5) M(-1) for PAVH+-SDS, and 1.5 x 10(3) to 3.0 x 10(3) M(-1) for the complexes of PAV0 with all three types of micelles. Using these data, an electrostatic potential difference on the micelle-water interface was calculated as 150 +/- 10 mV for CTAC, 140 +/- 10 mV for HPS and - 140 +/- 10 mV for SDS. The results suggest that PAV aromatic rings are located in the hydrophobic part of the micelle. The electrostatic attraction or repulsion of the protonated quinoline nitrogen and surfactant headgroups changes the affinity of PAV to micelles and, thus, shifts the ionization equilibrium of PAV. The electrostatic potential of HPS micellar surface is determined by the cationic dimethylammonium headgroup fragment, whereas the anionic sulfate fragment attenuates the effective charge of HPS headgroup.     

Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp

Three cellulases, one hemicellulase and three pectinases were used, separately or in binary and ternary combinations, to hydrolyze dried beet-pulp, a by-product of the sugar industry. By IE-HPLC the compositions and concentrations of the sugars released were determined. The results obtained by enzymatic saccharification were compared to those obtained by acid hydrolysis. The synergistic action of cellulolytic and pectinolytic enzymes in release of total monosaccharides, and of glucose, arabinose and galacturonic acid was also studied. The combination of cellulase, hemicellulase and pectinase, commercially available, was as effective in degrading the beet pulp as the acid hydrolysis. Pectinase appeared to be the most important enzyme, since by hydrolyzing the pectic surface of the lignocellulosic substrate, it favoured the degradation of cellulose and hemicellulose by the respective enzymes.     

Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Lipid Composition in Synaptic and Nonsynaptic Mitochondria from Rat Brains and Effect of Aging

The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.     

Basal metabolism and dynamic activity of food in obesity

Metabolic rate of obese patients is reported, recorded to cellular active mass. The metabolic rate is higher in obese patients and the value is diucthy related to individual actual weight. The dinamic-specific action of proteins is normal.     

Cliffs Used as Communal Roosts by Andean Condors Protect the Birds from Weather and Predators

The quality and availability of resources influence the geographical distribution of species. Social species need safe places to rest, meet, exchange information and obtain thermoregulatory benefits, but those places may also serve other important functions that have been overlooked in research. We use a large soaring bird that roosts communally in cliffs, the Andean condor (Vultur gryphus), as a model species to elucidate whether roost locations serve as a refuge from adverse weather conditions (climatic refuge hypothesis, CRH), and/or from predators or anthropogenic disturbances (threats refuge hypothesis, TRH). The CRH predicts that communal roosts will face in the opposite direction from where storms originate, and will be located in climatically stable, low precipitation areas. The TRH predicts that communal roosts will be large, poorly accessible cliffs, located far from human-made constructions. We surveyed cliffs used as communal roosts by condors in northwestern Patagonia, and compared them with alternative non-roosting cliffs to test these predictions at local and regional scales. We conclude that communal roosting places provide refuge against climate and disturbances such as, for instance, the threats of predators (including humans). Thus, it is not only the benefits gained from being aggregated per se, but the characteristics of the place selected for roosting that may both be essential for the survival of the species. This should be considered in management and conservation plans given the current scenario of global climate change and the increase in environmental disturbances.     

Virological consequences of early events following cell-cell contact between human immunodeficiency virus type 1-infected and uninfected CD4+ cells

Human immunodeficiency virus type 1 (HIV-1)-infected cells transmit viral products to uninfected CD4⁺ cells very rapidly. However, the natures of the transmitted viral products and the mechanism of transmission, as well as the relative virological consequences, have not yet been fully clarified. We studied the virological events occurring a few hours after contact between HIV-1-infected and uninfected CD4⁺ cells using a coculture cell system in which the virus expression in target cells could be monitored through the induction of a green fluorescent protein reporter gene driven by HIV-1 long terminal repeats. Within 16 h of coculture, we observed two phenomena not related to the cell-free virus infection, i.e., the formation of donor-target cell fusions and a fusion-independent internalization of viral particles likely occurring at least in part through intercellular connections. Both events depended on the expression of Env and CD4 in donor and target cells, respectively, whereas the HIV-1 internalization required clathrin activity in target cells. Importantly, both phenomena were also observed in cocultures of primary CD4⁺ lymphocytes, while primary macrophages supported only HIV-1 endocytosis. By investigating the virological consequences of these events, we noticed that while fused cells released infectious HIV-1 particles, albeit with reduced efficiency compared with donor cells, no virus expression was detectable upon HIV-1 endocytosis in target cells. In sum, the HIV-1 transmission following contact between an HIV-1-infected and an uninfected CD4⁺ cell can occur through different mechanisms, leading to distinguishable virological outcomes.