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Ocular hypertension is the greatest known risk factor for glaucoma that affects an estimated 70 million people worldwide. Lowering intraocular pressure (IOP) remains the mainstay of therapy in the management of glaucoma. By means of microarray analysis, we have discovered that 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)) regulates genes that are known to be involved in the determination of intraocular pressure (IOP). Topical administration of 1α,25-(OH)(2)D(3) or its analog, 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D(3) (2MD), markedly reduces IOP in non-human primates. The reduction in IOP is not the result of reduced aqueous humor formation, while a 35% increase in aqueous humor drainage by 1α,25-(OH)(2)D(3) was found but this increase did not achieve significance. Nevertheless, our results suggest that 1α,25-(OH)(2)D(3), or an analog thereof, may present a new approach to the treatment of glaucoma., (Copyright © 2011. Published by Elsevier Inc.)

The requirement for TRPV6 for vitamin D-dependent intestinal calcium absorption in vivo has been examined by using vitamin D-deficient TRPV6 null mice and littermate wild-type mice. Each of the vitamin D-deficient animals received each day for 4 days 50 ng of 1,25-dihydroyvitamin D(3) in 0.1 ml of 95% propylene glycol:5% ethanol vehicle or vehicle only. Both the wild-type and TRPV6 null mice responded equally well to 1,25-dihydroxyvitamin D(3) in increasing intestinal calcium absorption. These results, along with our microarray data, demonstrate that TRPV6 is not required for vitamin D-induced intestinal calcium absorption and may not carry out a significant role in this process. These and previous results using calbindin D9k null mutant mice illustrate that molecular events in the intestinal calcium absorption process in response to the active form of vitamin D remain to be defined.

The exact role of calbindin D9k in vitamin D-mediated calcium absorption has been debated but remains unsettled. In 129/OlaHsd mice, calbindin D9k was found highest in duodenum (36-50%) and kidney (24-34%) followed by stomach, lung and uterus. Age does not affect the relative distribution of calbindin D9k but it does decline with age in duodenum of both male and female 129/Ola mice. Recently, we produced a null calbindin D9k mutant 129/OlaHsd mouse; this mouse proved to be indistinguishable from the wild-type in phenotype and in a serum calcium level regardless of age or gender. We have now examined directly whether the mutant mouse can absorb calcium from the intestine in response to the active form of vitamin D. The calbindin D9k null mutant mouse is fully able to absorb calcium from the intestine in response to 1,25-dihydroxyvitamin D3. It is, therefore, clear that calbindin D9k is not required for vitamin D-induced intestinal calcium absorption.

1Alpha,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), the biologically active form of vitamin D(3), not only plays a major role in mammalian calcium and phosphorous homeostasis but also exerts pleiotropic effects on cell proliferation, differentiation and the immune system. Further, vitamin D is believed to play a significant role in the prevention of colon, prostate, and breast cancer and in reducing the risk of autoimmune diseases. To gain insight into the mechanism whereby vitamin D can have such diverse actions, we have employed microarray technology. We studied the effect of a single dose of 1,25-(OH)(2)D(3) on gene expression in the intestine of vitamin D-deficient rats. Within 6 h, 1,25-(OH)(2)D(3) stimulates the expression of several phase I and phase II biotransformation genes. There is also an increased expression of antioxidant genes. These results support the idea that vitamin D is a significant factor in detoxification and protection against environmental toxins.

Since the discovery of calbindin D(9k), its role in intestinal calcium absorption has remained unsettled. Further, a wide distribution of calbindin D(9k) among tissues has argued for its biological importance. We discovered a frameshift deletion in the calbindin D(9k) gene in an ES cell line, E14.1, that originated from 129/OlaHsd mice. We produced mice with the mutant calbindin D(9k) gene by injecting the E14.1 ES cell subline into the C57BL/6 host blastocysts and proved that these mice lack calbindin D(9k) protein. Calbindin D(9k) knockout mice were indistinguishable from wild-type mice in phenotype, were able to reproduce, and had normal serum calcium levels. Thus, calbindin D(9k) is not required for viability, reproduction, or calcium homeostasis.

Microarray technology has been used to discover 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) induced gene expression changes in rat small intestine in vivo. Here, we report gene expression changes related to intestinal absorption or transport, the immune system and angiogenesis in response to 1,25-(OH)(2)D(3). Vitamin D deficient rats were intrajugularly given vehicle or vehicle containing 730 ng of 1,25-(OH)(2)D(3)/kg of body weight. Intestinal mRNA was harvested from duodenal mucosa at 15 min, 1, 3, and 6 h post-injection and studied by Affymetrix microarrays. Genes significantly affected by 1,25-(OH)(2)D(3) were confirmed by quantitative RT-PCR with remarkable agreement. The most strongly affected gene in intestine was CYP24 with 97-fold increase at 6 h post-1,25-(OH)(2)D(3) treatment. Intestinal calcium absorption genes: TRPV5, TRPV6, calbindin D(9k), and Ca(2+) dependent ATPase all were up-regulated in response to 1,25-(OH)(2)D(3), supporting the currently accepted mechanism of 1,25-(OH)(2)D(3) induced transcellular calcium transport. However, a 1,25-(OH)(2)D(3) suppression of several intra-/intercellular matrix modeling proteins such as sodium/potassium ATPase, claudin 3, aquaporin 8, cadherin 17, and RhoA suggests a vitamin D regulation of tight junction permeability and paracellular calcium transport. Several other genes related to the immune system and angiogenesis whose expression was changed in response to 1,25-(OH)(2)D(3) provided evidence for an immunomodulatory and anti-angiogenic role of 1,25-(OH)(2)D(3).

Diphosphoryl lipid A derived from the nontoxic LPS of Rhodobacter sphaeroides (RsDPLA) has been shown to be a powerful LPS antagonist in both human and murine cell lines. In addition, RsDPLA also can protect mice against the lethal effects of toxic LPS. In this study, we complexed both the deep rough LPS from Escherichia coli D31 m4 (ReLPS) and RsDPLA with 5- and 30-nm colloidal gold and compared their binding to the RAW 264.7 cell line by electron microscopy. Both ReLPS and RsDPLA bound to the cells with the following observations. First, binding studies revealed that pretreatment with RsDPLA completely blocked the binding and thus internalization of ReLPS-gold conjugates to these cells at both 37 degrees C and 4 degrees C. Second, ReLPS was internalized via micropinocytosis (noncoated plasma membrane invaginations) involving formation of caveolae-like structures and leading to the formation of micropinocytotic vesicles, macropinocytosis (or phagocytosis), formation of clathrin-coated pits (receptor mediated), and penetration through plasma membrane into cytoplasm. Third, in contrast, RsDPLA was internalized predominantly via macropinocytosis. These studies show for the first time that RsDPLA blocks the binding and thus internalization of LPS as observed by scanning and transmission electron microscopy.

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence of N-ethylamide of o-sulfobenzoylacetic acid (EASBA) at pH 5.0-7.5. In the oxidation of o-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases both K(m) and Vm in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.

Physico-chemical properties of the recombinant L. mingrelica luciferase synthesized by E. coli cells have been studied. The catalytic and spectral properties of recombinant luciferase were similar to those of the native enzyme but the former was less stable in the presence of the additional Cys residue. The mutant forms of L. mingrelica firefly luciferase with point mutations Cys-82-->Ala, Cys-260-->Ala, Cys-393-->Ala and Thr-204-->Asp, have been constructed using the method of site-specific mutagenesis. Mutations Cys-82,260,393-->Ala changed slightly the Km values for ATP and luciferin but did not influence kcat. The Cys-393-->Ala mutant appeared to be more stable in comparison with the native enzyme. Mutation Thr-204-->Asp resulted in a 8-fold increase in the ATP binding constant and in a 2-fold increase in the kcat, thus indicating that Thr-204 may be located in the ATP-binding region of luciferase. Dithiothreitol, ethylene glycol, bovine serum albumin and trehalose had a stabilizing effect on the native, recombinant and mutant luciferases.

The Photinus pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E. coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements. The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C. The maximal amount of the Ph. pyralis protein reached 4.5%, while that of L. mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation. The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction. The increase in the enzyme activity correlated well with the increase in the ATP content in the cell. The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation. This transformation is considerably behind the protein biosynthesis process. Intracellular metabolic reactions have been shown to play an active part in these conformational changes.

The Photinus pyralis and Luciola mingrelica luciferases genes expression kinetics has been studied, repectively, on the pME61 and pJG lambda plasmids with a thermoinducible lambda PR promoter in the E. coli strain CA under conditions of a pH shift. An increase in the enzyme activity after a pH shift has been revealed. The maximal amount of the firefly Ph. pyralis luciferase reaches 5.3%, while the maximal amount of the L. mingrelica enzyme reaches 4.9% of the total amount of cellular proteins in case of pHout shift to 9.0 according to the SDS gel electrophoresis data; that in case of a pHout shift to 8.5 is 4.9% and 4.5%, respectively. The enzyme activation correlates well with the dynamics of pHout and pHin.

Recent data from gene engineering, kinetic and fluorescent studies concerning the structure and functions of firefly luciferase are reviewed. Some new trends in the development of bioluminescent methods of control over bacterial contaminations, dynamics of intracellular processes and methods of bioluminescent detection in immuno- and DNA-assays are described. Possible applications of luciferase genes as markers are considered.

We have cloned cDNA encoding luciferase in Luciola mingrelica, fireflies living near the Black Sea in southern Russia, and obtained high level expression of the cloned sequences in Escherichia coli. The nucleotide sequences of two isolated clones were determined; five single base differences were observed, but none resulted in a change in the encoded amino acid residue. The cDNA encoded a protein of 548 amino acid residues. The overall amino acid sequence identity with the luciferase from Photinus pyralis, the North American firefly, was 67%, while comparison of the L. mingrelica luciferase with L. cruciata and L. lateralis, both indigenous to Japan, showed about 80% of the residues were strictly conserved. A novel overexpression system which employs the regulatory genes of the luminous bacterium Vibrio fischeri allowed growth of cultures to high cell density and high luciferase content, facilitating purification of the enzyme. Luciferase was purified to homogeneity in good yield from lysates of recombinant E. coli by ammonium sulfate fractionation and chromatography on columns of DEAE Sephadex and Blue Sepharose. The physicochemical properties of the luciferases from the available recombinant sources are significantly different and should allow detailed investigations into the mechanism of the bioluminescence reaction and the physical basis of the differences in the color of light emitted from the various enzymes.

Poly (A+)RNA was isolated from the lanterns of adult fireflies of L. mingrelica. The poly (A+)RNA was translated in a cell-free translation mixture from rabbit reticulocyte and from Krebs II mouse ascites cells and in Xenopus laevis frog oocytes. The synthesis of the enzymatically active firefly luciferase was demonstrated in all systems. In the cell-free systems a maximal quantity of luciferase is synthesized during the first 1-2 h, then the decreasing activity of luciferase is observed. The optimal concentration of mRNA for translation in the mouse ascites cell extract was found. The kinetics of the synthesis of the luciferase, its pathway and stability in X. laevis oocytes was studied. It was observed that luciferase is secreted from oocytes into the incubation medium.