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Since the discovery of calbindin D9k, 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 129͞OlaHsd mice. We produced mice with the mutant calbindin D9k gene by injecting the E14.1 ES cell subline into the C57BL͞6 host blastocysts and proved that these mice lack calbindin D 9k protein. Calbindin D9k 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.vitamin D 3 ͉ E14ES cells ͉ calcium homeostasis F or decades, a major physiologic function of calbindin D 9k was believed to be a carrier of calcium during intestinal calcium absorption (1). However, the detailed mechanism of vitamin D-induced intestinal calcium absorption still is not fully understood. According to the currently accepted model, vitamin D-mediated transcellular calcium absorption in the intestine proceeds through the calcium channel proteins TRPV5 and TRPV6 (2) with the involvement of cellular calcium transfer protein calbindin D 9k (1, 3) and a calcium extrusion protein, calcium ATPase (PMCA 1b ) (4). The data on regulation of epithelial calcium channels (TRPV5 and TRPV6), calbindin D 9k , and PMCA 1b expression by 1,25-dihydroxyvitamin D 3 seem clear, and these genes have vitamin D responsive elements in their promoter regions (5-7).Calbindin D 9k was first discovered as the mammalian counterpart to the calbindin D 28k that was found in chick duodenal mucosa in response to vitamin D 3 (8, 9). Later, calbindin D 9k from vitamin D 3 -responsive rat intestinal mucosa was identified, purified, and characterized (9, 10). Since its discovery in 1967, the role of calbindin D 9k protein in vitamin D 3 -mediated intestinal calcium absorption has been intensively studied but still remains unsettled. In the meantime, a wide distribution of this protein in tissues not involved in calcium absorption has been noted, arguing for its importance in biology (11-13).In 1969, Harmeyer and DeLuca (14) Results and DiscussionDiscovery of the Mutant Mouse Calbindin D9k Gene. The P1 clone 7681 with an 85-kb insert of genomic DNA͞SauIIIA containing the mouse calbindin D 9k gene was purchased from Incyte Genomics (Palo Alto, CA). The P1 mouse library was generated by Sternberg et al. (20) by using genomic DNA from the ES cell line subclone E14.1, which is derived from E14 ES cells (21). The ES cell line E14 was derived from the inbred mouse strain 129͞OlaHsd in 1985 by Hooper et al. (22). The insert was cut with BamHI and subcloned into pBluescript (Stratagene, La Jolla, CA). The clone that was positive for the calbindin D 9k gene was selected by Northern blot analysis, and a plasmid with the mutant calbindin D 9k insert was isolated. We have sequenced the 10.409-kb insert containi...

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S-nitrosoglutathione (GSNO, 50 M) inhibited the initial rate of thrombin-catalyzed human and bovine fibrinogen polymerization by Ϸ50% to 68% respectively. Inhibition was also observed with other structurally varied S-nitrosothiols (RSNOs) including sugar derivatives of S-nitroso-N-acetylpenicillamine (SNAP). The fact that the same concentration of GSNO had no effect on thrombindependent hydrolysis of tosylglycylprolylarginine-4-nitroanilide acetate suggested that this inhibition was due to GSNO-induced changes in fibrinogen structure. This result was confirmed by CD spectroscopy where GSNO or S-nitrosohomocysteine increased the ␣-helical content of fibrinogen by Ϸ15% and 11%, respectively. S-carboxymethylamido derivatives of glutathione or homocysteine had no effect on the fibrinogen secondary structure. The GSNO-dependent secondary structural effects were reversed on gel filtration chromatography, suggesting that the effects were allosteric. Further evidence for fibrinogen-GSNO interactions was obtained from GSNO-dependent quenching of the intrinsic fibrinogen Trp fluorescence and the perturbation of the GSNO circular dichroic absorbance as a function of [fibrinogen]. The K ds of 3 to 10 M for fibrinogen-GSNO interactions with a stoichiometry of 2:1 (GSNO:fibrinogen) were estimated from isothermal titration calorimetry and fluorescence quenching, respectively. These results suggest that RSNOs induce changes to fibrinogen structure by interacting at specific aromatic rich domains. Three such putative RSNO-binding domains have been identified in the unordered, aromatic residue-rich C-termini of the ␣-chains of fibrinogen.

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Akhter S, Zhang Z, Jin JP. The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I. Am J Physiol Heart Circ Physiol 302: H923-H933, 2012. First published December 2, 2011; doi:10.1152/ajpheart.00637.2011.-In addition to the core structure conserved in all troponin I isoforms, cardiac troponin I (cTnI) has an ϳ30 amino acids NH 2-terminal extension. This peptide segment is a heart-specific regulatory structure containing two Ser residues that are substrates of PKA. Under ␤-adrenergic regulation, phosphorylation of cTnI in the NH 2-terminal extension increases the rate of myocardial relaxation. The NH 2-terminal extension of cTnI is also removable by restrictive proteolysis to produce functional adaptation to hemodynamic stresses. The molecular mechanism for the NH 2-terminal modifications to regulate the function of cTnI is not fully understood. In the present study, we tested a hypothesis that the NH 2-terminal extension functions by modulating the conformation of other regions of cTnI. Monoclonal antibody epitope analysis and protein binding experiments demonstrated that deletion of the NH 2-terminal segment altered epitopic conformation in the middle, but not COOH-terminal, region of cTnI. PKA phosphorylation produced similar effects. This targeted longrange conformational modulation corresponded to changes in the binding affinities of cTnI for troponin T and for troponin C in a Ca 2ϩ -dependent manner. The data suggest that the NH2-terminal extension of cTnI regulates cardiac muscle function through modulating molecular conformation and function of the core structure of cTnI.troponin I phosphorylation; proteolytic NH 2-terminal truncation; TnITnT interface; cardiac muscle regulation; protein conformation analysis THE CONTRACTION OF CARDIAC muscle is regulated by binding of cytosolic Ca 2ϩ to troponin, which activates cross bridge cycling between sarcomeric myosin and actin filaments. The troponin complex consists of three protein subunits: the Ca 2ϩ -binding subunit troponin C (TnC), the tropomyosin-binding subunit troponin T (TnT), and the inhibitory subunit troponin I (TnI) (19). The function of TnI is essential to cardiac muscle contraction (34).Three homologous genes are present in vertebrate species encoding the fast skeletal muscle, slow skeletal muscle, and cardiac isoforms of TnI (20,29). Cardiac TnI (cTnI) is the newest member evolved in the family of TnI isoform genes (7). In addition to the ϳ180 amino acids core structure that is highly conserved in the three TnI isoforms in all vertebrates, cTnI has a unique NH 2 -terminal extension of ϳ30 amino acids, which is not found in the two skeletal muscle TnI isoforms.Showing functional conservation and exchangeability among the TnI isoforms, embryonic cardiac muscle utilizes solely slow skeletal muscle TnI. During development, it is completely replaced by cTnI in the adult heart (22, 36). Therefore, the NH 2 -terminal extension of cTnI is not an essential structure for the basic contractility of c...

Summary The troponin complex plays an essential role in the thin filament regulation of striated muscle contraction. Of the three subunits of troponin, troponin I (TnI) is the actomyosin ATPase inhibitory subunit and its effect is released upon Ca2+-binding to troponin C. The exon 8-encoded COOH-terminal end segment represented by the last 24 amino acids of cardiac TnI is highly conserved and critical to the inhibitory function of troponin. Here we investigated the function and calcium regulation of the COOH-terminal end segment of TnI. A TnI model molecule was labeled with Alexa fluor 532 at a Cys engineered at the COOH-terminal end and used to reconstitute tertiary troponin complex. A Ca2+-regulated conformational change in the COOH terminus of TnI was shown by a sigmoid-shape fluorescence intensity titration curve similar to that of the circular dichroism calcium titration curve of troponin C. Such corresponding Ca2+-responses are consistent with the function of troponin as a coordinated molecular switch. Reconstituted troponin complex containing a mini-troponin T lacking its two tropomyosin-binding sites showed a saturable binding to tropomyosin at pCa 9 but not at pCa 4. This Ca2+-regulated binding was diminished when the COOH-terminal 19 amino acids of cardiac TnI were removed. These results provided novel evidence for suggesting that the COOH-terminal end segment of TnI participates in the Ca2+-regulation of muscle thin filament through an interaction with tropomyosin.

Background: Unsafe food consumption is a severe problem because of heavy metal contamination, which is caused by director indirect activities of industries. The present study was conducted to assess the risk of human health by Heavy metals (Cu, Co, Fe, Zn and Mn) through the intake of vegetables and fishes obtained from the area adjacent to the Hazaribag tannery campus, Dhaka, Bangladesh. Result: The trend of mean metal concentration in Buriganga river water was Fe >Mn > Zn > Cu > Co and according to Department of Environment, Dhaka Bangladesh (DoE) (1999) the value of the above metals are within the permissible limit of irrigation water except Fe. An assessment of risk involved due to consumption of contaminated food also calculated. The trend of metals in vegetables was Fe > Mn > Zn > Cu > Co and in fishes the trend was Fe > Zn >Mn > Co > Cu. Accumulation of trace elements in vegetables was lower than maximum tolerable levels proposed by FAO/WHO food standard programme (2001) with the exception of Fe and Co respectively. In fishes metal concentration was lower than safe limit set by WHO (1989) except Mn. The Metal Pollution Index (MPI) for all the foodstuffs showed a higher value, however the calculated Health Risk Index (HRI) indicated no risk to human health upon consumption of those foodstuffs. Conclusion: The overall study suggests that foodstuff in the area were contaminated by the assayed metals and long-term consumption can cause potential health risks to consumers.