Sol-gel encapsulation of lactate dehydrogenase for optical sensing of L-lactate

Sol-gel encapsulation of lactate dehydrogenase and its cofactor can be employed as a disposable sensor for L-lactate. The sensor utilized the changes in absorbance or fluorescence from the reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. Although, problems such as diminished enzymatic activity and/or leaching of enzyme from the sol-gel matrix occurred, the sol-gel process is sufficiently mild to permit retention of enzymatic activity. The apparent activity of LDH in the sensor is at least 10% of that of the dissolved enzyme. The sensor has a linear dynamic range over the normal physiological L-lactate level and has a long-term storage stability of at least 3 weeks.     

Ricardo Miledi and the calcium hypothesis of neurotransmitter release

Ricardo Miledi has made significant contributions to our basic understanding of how synapses work. Here I discuss aspects of Miledi's research that helped to establish the requirement of presynaptic calcium for neurotransmitter release, from his earliest scientific studies to his classic experiments in the squid giant synapse.     

Solution structure of the DNA-binding domain of interleukin enhancer binding factor 1 (FOXK1a)

Interleukin enhancer binding factor (ILF) binds to the interleukin-2 (IL-2) promoter and regulates IL-2 gene expression. In this study, the 3D structure of the DNA-binding domain of ILF was determined by multidimensional NMR spectroscopy. NMR structure analysis revealed that the DNA-binding domain of ILF is a new member of the winged helix/forkhead family, and that its wing 2 contains an extra alpha-helix. This is the first study to report the presence of a C-terminal alpha-helix in place of a typical wing 2 in a member of this family. This structural difference may be responsible for the different DNA-binding specificity of ILF compared to other winged helix/forkhead proteins. Our deletion studies of the fragments of ILF also suggest that the C-terminal region plays a regulatory role in DNA binding.     

Antibiotic activity of lectins from marine algae against marine vibrios

Saline and aqueous ethanol extracts of marine algae and the lectins from two red algal species were assayed for their antibiotic activity against marine vibrios. Experimental studies were also carried out on the influence of environmental factors on such activity, using batch cultures. The results indicated that many of the saline extracts of the algal species were active and that the activity was selective against those vibrios assayed. The algal extracts were active against Vibrio pelagius and the fish pathogen V. vulnificus, but inactive against V. neresis. Algal lectins from Eucheuma serra (ESA) and Galaxaura marginata (GMA) strongly inhibited V. vulnificus but were inactive against the other two vibrios. The antibacterial activity of algal extracts was inhibited by pretreatment with various sugars and glycoprotein. Extracts of the two red algae, E. serra and Pterocladia capillacea, in saline and aqueous ethanol, inhibited markedly the growth rate of V. vulnificus at very low concentrations. Culture results indicated that metabolites active against V. vulnificus were invariably produced in P. capillacea over a wide range of temperature, light intensity, and nutritional conditions. Enhanced antibacterial activity occurred when P. capillacea was grown under higher irradiance, severe nutrient stress and moderate temperature (20 °C), reflecting the specific antibiotic characteristics of this alga. The strong antibiotic activity of lectins towards fish pathogenic bacteria reveals one of the important roles played by algal lectins, as well as the potential high economic value of those marine algae assayed for aquaculture and for biomedical purposes.     

Involvement of hydrogen peroxide and nitric oxide in expression of the ipomoelin gene from sweet potato

The IPO (ipomoelin) gene was isolated from sweet potato (Ipomoea batatas cv Tainung 57) and used as a molecular probe to investigate its regulation by hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) after sweet potato was wounded. The expression of the IPO gene was stimulated by H(2)O(2) whether or not the plant was wounded, but its expression after wounding was totally suppressed by the presence of diphenylene iodonium, an inhibitor of NADPH oxidase, both in the local and systemic leaves of sweet potato. These results imply that a signal transduction resulting from the mechanical wounding of sweet potato may involve NADPH oxidase, which produces endogenous H(2)O(2) to stimulate the expression of the IPO gene. The production of H(2)O(2) was also required for methyl jasmonate to stimulate the IPO gene expression. On the contrary, NO delayed the expression of the IPO gene, whereas N(G)-monomethyl-L-arginine monoacetate, an inhibitor of NO synthase, enhanced the expression of the IPO gene after the plant was wounded. This study also demonstrates that the production of H(2)O(2) stained with 3,3'-diaminobenzidine hydrochloride could be stimulated by wounding but was suppressed in the presence of NO. Meanwhile, the generation of NO was visualized by confocal scanning microscope in the presence of 4,5-diaminofluorescein diacetate after sweet potato was wounded. In conclusion, when sweet potato was wounded, both H(2)O(2) and NO were produced to modulate the plant's defense system. Together, H(2)O(2) and NO regulate the expression of the IPO gene, and their interaction might further stimulate plants to protect themselves from invasions by pathogens and herbivores.     

Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton

A family of nine Salmonella typhimurium type III secretion effectors with a conserved amino-terminus have been defined. Three family members (SifA, SifB and SseJ) have previously been demonstrated to localize to the Salmonella-containing vacuole and to Salmonella-induced filaments. In contrast, we demonstrate that two other family members, SspH2 and SseI, co-localized with the polymerizing actin cytoskeleton. These proteins also interacted with the mammalian actin cross-linking protein filamin in the yeast two-hybrid assay through their highly conserved amino-terminal domains. This amino-terminus was sufficient to direct localization to the polymerizing actin cytoskeleton, suggesting that the interaction with filamin is important for this subcellular localization. In addition, SspH2 co-localized with vacuole-associated actin polymerizations (VAP) induced by intracellular bacteria through the Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS). SspH2 interacted with the actin-binding protein profilin in the yeast two-hybrid assay and by affinity chromatography. This interaction was highly specific to SspH2 and was mediated by its carboxy-terminus. Furthermore, SspH2 inhibited the rate of actin polymerization in vitro, suggesting that it functions to reduce or remodel VAP. Strains with mutations in sspH2 and sseI retained the ability to form VAP. However, a third intracellular virulence factor, spvB, which ADP-ribosylates actin, strongly inhibited VAP formation in HeLa cells, suggesting a more subtle effect for SspH2 and SseI on the actin cytoskeleton.     

A peritoneal mesothelioma in a captive aardwolf (Proteles cristatus)

A 10-year-old male aardwolf (Proteles cristatus) was presented abdominal distention and emaciation for 3 months. Physical examination revealed firm abdominal masses with effusions. Cytologic assessment of the effusion showed uniform round tumor cells with a surface brush border. Necropsy showed white velvety masses covering the peritoneal surface of the liver, gall bladder, stomach, omentum, mesentery, spleen, intestine, abdominal wall and diaphragm. Histologic examination demonstrated papillary projections, lined with cuboidal tumor cells supported by fibrous connective tissue cores, arising from the serosa of visceral organs. Cytoplasmic vacuolation and a surface brush border were evident on some cells under light microscopy. Tumor cells stained positive for both cytokeratin (AE1/AE3) and vimentin. Electron microscopy showed prominent surface microvilli, rough endoplasmic reticulum, mitochondria and desmosomes in tumor cells. This may be the first reported case of peritoneal mesothelioma in a captive wild aardwolf.