Characterization of two low Em forms of cytochrome a3 and their carbon monoxide complexes in mammalian cytochrome c oxidase.

Evidence is presented for the existence of two forms of low-potential cytochrome a3. One appears to be similar to the low-spin form reported by Nicholls, P., and V. Hildebrandt (1978 Biochem. J. 173:65-72) and Wrigglesworth, J. M., J. Elsden, A. Chapman, N. Van der Water, and M. F. Grahn (1988. Biochim. Biophys. Acta. 936:452-464). It has a reduced Soret peak near 428 nm and a prominent alpha peak near 602 nm. This form is seen when the enzyme is either supplemented with lipoprotein or incorporated into a liposomal membrane, preexposed to a voltage greater than 400 mV for at least 30 min, and titrated in the presence of approximately 1 mM K3Fe(CN)6. The other form has a reduced Soret peak near 446 nm, and no prominent alpha peak. The 428-nm form has an Em near 175 mV and forms a CO complex with an Em near 225 mV. The 446-nm form has an Em near 200 mV and forms a CO complex with an Em near 335 mV.     

Pilot scale production of functional foods using red palm olein: Antioxidant,vitamins’ stability and sensory quality during storage

The objective of this research work was to produce acceptable quality functional foods, namely, extruded snacks, digestive biscuits and pan bread, on a pilot scale, using vitamin E and β-carotene-rich red palm olein (RPOL) and red palm shortening (RPS). These products were evaluated for their chemical composition and sensory quality along with the antioxidants and vitamin contents during the six months of storage at room temperature (22 ± 1 °C). Extruded snacks and digestive biscuits prepared with RPOL and RPS were found to be good sources of these antioxidant vitamins. The average β-carotene content of the control and test snacks at the end of six months of storage ranged from 26.8 to 56.1 mg/kg fat, and from 430.9 to 468.9 mg/kg fat, respectively. The total vitamin E content in control and test snacks made in Plant No. 1 decreased after six months of storage from 786.1 to 704.4 mg/kg fat, and from 765.1 to 695.4 mg/kg fat, respectively. As expected, the total tocotrienol content was four to five times higher than the total tocopherols in control biscuits. The RPOL containing 600–750 ppm of carotenes (mainly α- and β-carotenes), 710–774 ppm of vitamin E, was found to be suitable for industrial application in producing acceptable quality pan bread, digestive biscuits and snacks. These functional foods contained significant amounts of β-carotene and total vitamin E, indicating the possibility of producing such foods rich in these two of the important antioxidant vitamins coming from a natural source. The research findings strongly indicate that good-quality pan bread, extruded snacks and digestive biscuits can successfully be produced to offer healthier eating choices to the consumers of this region, thereby promoting better health and productivity among the population.     

Bacterial disinfectant resistance—a challenge for the food industry

The focus on hygiene in the food industry has resulted in an increasing use of chemical disinfection and it has been speculated that this will impose a selective pressure and contribute to the emergence of disinfectant-resistant microorganisms. The frequency of strains with a low-level resistance to quaternary ammonium compounds (QAC) is relatively high for Listeria monocytogenes (10%), Staphylococcus spp. (13%) and Pseudomonas spp. (30%) and lower for lactic acid bacteria (1.5%) and coliforms (1%) isolated from food and food processing industry. In general, bacteria isolated after disinfection are more resistant and represent a potential food safety or food spoilage problem. Adaptation to disinfectants may be accompanied by cross-resistance to related disinfectants. We have recently found a genetic linkage between resistance to QAC and antibiotics in food associated staphylococci, and there is a growing concern about cross-resistance between antibiotics and disinfectants.Disinfectant resistance can in most cases be prevented by effective cleaning and disinfection procedures. More effort should be made to avoid build-up of resistance in food production environments.     

Seimatoantlerium tepuiense gen. nov., a unique epiphytic fungus producing taxol from the Venezuelan Guyana.

Seimatoantlerium gen. nov., type species, S. tepuiense sp. nov. is proposed for an acervular fungus producing 4-septate, holoblastic conidia with 6-8 unbranched, apical appendages that dehisce as an appendage apparatus and also commonly possessing one or two exogenous basal appendages as well as a pedicel. It is compared with Seimatosporium, Seimatosporiopsis, and other genera. It is epiphytic on Maguireothamnus speciosus, a rubiaceous plant endemic to the tepuis of southeastern Venezuela. It produces the anti-oomycetous anticancer compound, taxol, as shown by immunological and spectroscopic methods. Taxol production is discussed relative to the ability of this fungus to exist in an extremely moist ecosystem, as well as to its relationship to other plant associated fungi.     

Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets

Ral GTPases may be involved in calcium/calmodulin-mediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calcium-independent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.     

A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.