Physiologic diversity and development of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate numerous nonvisual phenomena, including entrainment of the circadian clock to light-dark cycles, pupillary light responsiveness, and light-regulated hormone release. We have applied multielectrode array recording to characterize murine ipRGCs. We find that all ipRGC photosensitivity is melanopsin dependent. At least three populations of ipRGCs are present in the postnatal day 8 (P8) murine retina: slow onset, sensitive, fast off (type I); slow onset, insensitive, slow off (type II); and rapid onset, sensitive, very slow off (type III). Recordings from adult rd/rd retinas reveal cells comparable to postnatal types II and III. Recordings from early postnatal retinas demonstrate intrinsic light responses from P0. Early light responses are transient and insensitive but by P6 show increased photosensitivity and persistence. These results demonstrate that ipRGCs are the first light-sensitive cells in the retina and suggest previously unappreciated diversity in this cell population.     

Regional physiological adaptation of the central nervous system deiodinases to iodine deficiency

The goal of the present investigation was to analyze the types 2 (D2) and 3 (D3) iodothyronine deiodinases in various structures within the central nervous system (CNS) in response to iodine deficiency. After 5-6 wk of low-iodine diet (LID) or LID + 2 microg potassium iodide/ml (LID + KI; control), rats' brains were processed for in situ hybridization histochemistry for D2 and D3 mRNA or dissected, frozen in liquid nitrogen, and processed for D2 and D3 activities. LID did not affect weight gain or serum triiodothyronine, but plasma thyroxine (T4) was undetectable. In the LID + KI animals, D3 activities were highest in the cerebral cortex (CO) and hippocampus (HI), followed by the olfactory bulb and was lowest in cerebellum (CE). Iodine deficiency decreased D3 mRNA expression in all CNS regions, and these changes were accompanied by three- to eightfold decreases in D3 activity. In control animals, D2 activity in the medial basal hypothalamus (MBH) was similar to that in pituitary gland. Of the CNS D2-expressing regions analyzed, the two most responsive to iodine deficiency were the CO and HI, in which an approximately 20-fold increase in D2 activity occurred. Other regions, i.e., CE, lateral hypothalamus, MBH, and pituitary gland, showed smaller increases. The distribution of and changes in D2 mRNA were similar to those of D2 activity. Our results indicate that decreases in the expression of D3 and increases in D2 are an integral peripheral component of the physiological response of the CNS to iodine deficiency.     

Characterization of hyaluronidase isolated from Agkistrodon contortrix contortrix (Southern Copperhead) venom

Snake venoms are a rich source of enzymes including many hydrolytic enzymes. Some enzymes such as phospholipase A2, proteolytic enzymes, and phosphodiesterases are well characterized. However many enzymes, such as the glycosidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine which venom was the best source for isolation of the enzyme, the hyaluronidase activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highest activity, this venom was used for purification of hyaluronidase. Molecular weight was determined by matrix-assisted laser desorption ionization mass spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 61,000 Da. Substrate specificity studies indicated that the snake venom enzyme was specific only for hyaluronan and did not hydrolyze similar polysaccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin. The enzyme is an endo-glycosidase without exo-glycosidase activity, as it did not hydrolyze p-nitrophenyl-beta-D-glucuronide or p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. The cleavage point is at the beta1,4-glycosidic linkage and not at the beta1,3-glycosidic linkage. Thus, snake venom hyaluronidase is an endo-beta-N-acetylhexosaminidase specific for hyaluronan.     

Identification and kinetic analysis of the interaction between Nck-2 and DOCK180

Nck-2 is a newly identified adapter protein comprising three N-terminal SH3 domains and one C-terminal SH2 domain. We have identified in a yeast two-hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck-2. Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C-terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck-2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck-2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding. A major Nck-2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819-1836 of DOCK180. Two additional, albeit much weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and 1835-1852 respectively. Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15+/-1.9x10(-7) M and 3.24+/-1.9x10(-9) M mediate the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region of DOCK180. These studies identify a novel interaction between Nck-2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.     

Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4–6 h at 37°C. Journal of Industrial Microbiology & Biotechnology (2001) 26, 345–349. Received 30 August 2000/ Accepted in revised form 17 April 2001     

1,2,5-Thiadiazolidin-3-one 1,1 dioxide: a powerful scaffold for probing the S' subsites of (chymo)trypsin-like serine proteases

The 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I) embodies a motif that allows it to dock to the active site of (chymo)trypsin-like proteases in a predictable and substrate-like fashion. Consequently, inhibitors derived from this heterocyclic scaffold interact with both the S and S' subsites of an enzyme. Exploitation of binding interactions with both the S and S' subsites of a target enzyme may lead to compounds with greatly enhanced enzyme selectivity and inhibitory potency. This preliminary report describes the use of a series of compounds having the heterocyclic scaffold linked to various amino acids to probe the S' subsites of human leukocyte elastase (HLE), proteinase 3 (PR 3), and cathepsin G (Cat G). For comparative purposes, a series of compounds derived from a related scaffold, isothiazolidin-3-one 1,1 dioxide (II), was also generated. Several of the compounds were found to be highly potent and selective time-dependent inhibitors of HLE, PR 3, and Cat G.     

Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I

Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared. The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme. The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks. In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C. The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme. The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined. FRG/FRase I followed a ping-pong kinetic mechanism. It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis. For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate. FAD and riboflavin were also active substrates. FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively. All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I. Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP.