Understanding the temporal and spatial changes in calcium concentration has been a difficult endeavor for many years due to the relatively small changes in calcium concentration during messenging events, the rapid changes upon physiological messenging, and the unavailability of fast, efficient, and sensitive sensors to detect calcium changes. In addition, the key factors in calcium binding have yet to be determined due to the metal-metal interactions, cooperativity, and conformational change involved in calcium binding to natural calcium-binding proteins. To overcome these obstacles and to engineer calcium sensors for in vivo studies of calcium signaling events, calcium binding sites have been engineered into Green Fluorescent Protein. The engineered binding sites demonstrate terbium binding affinity from 2-30 ƒÝM and calcium binding affinity from 50-100 ƒÝM. Site 177 demonstrates green fluorescence when expressed in mammalian cells and produces a response to calcium concentration changes when expressed in the cytosol. Addition of the cycle 3 mutations (M153T, V163A, F99S) to Site 177 allowed for increased brightness in the emission of the chromophore but still exhibited calcium response. The second generation Site 1 demonstrates fluorescence response to calcium concentration changes when expressed both in the cytosol and in the endoplasmic reticulum. Addition of M153T and V163A to Site 1 allowed for expression of fluorescent protein at 37 ¢XC in HeLa cells and at 30 ¢XC in bacteria. Site 1-M153T/V163A exhibits chromophore fluorescence response to calcium with a Kd of 100 ƒÝM and competition with Rhodamine-5N produced a calcium Kd of 107 ƒÝM. This designed sensor, Site 1-M153T/V163A is the first demonstration of a designed calcium binding GFP with calcium response measured both in vivo and in vitro.

The chloride-induced corrosion of reinforcing steel in concrete structures has become a widespread durability problem throughout the world. When concrete structures come in contact with chloride sources, the chloride ions will diffuse through the body of the concrete and ultimately reach the steel. Not all of the chloride ions which penetrate the concrete remain free in the pore solution. Some of the ions become bound to the hydration products in a chemical reaction to form calcium chloroaluminate hydrate (Friedel' salt). It is also well known that only the portion of the chloride ions that remains free is responsible for causing damage to the concrete structures by corroding steel rebar. Thus, the chloride binding capacity of the cementitious matrix plays a major role in controlling chlorides ingress and, consequently, the corrosion of steel reinforcement in concrete. The chloride binding capacity is affected by cement composition, environmental factors, and by the source of the chlorides ( vs. ). To quantify the durability of new and existing structures, a clear understanding of the mechanisms of chloride penetration into the concrete cover is required.<p> Currently, most of the models available in the published literature for calculating free chloride ions in concrete use Ficks law for chloride transport and chloride binding isotherms to account for bound chlorides. Binding isotherms are cement and environment specific. Thus, the existing models cannot be used for all types of cement and variable general environmental exposure conditions such as temperatures, pH levels, and chloride sources. A general mechanistic approach that can overcome those limitations is proposed in this thesis based on the concepts of ion-exchange theory for an accurate determination of chloride ingress in concrete under variable environmental conditions.<p> Some of the model input parameters, such as exchange capacity and the equilibrium constant for the exchange reaction, were not easy to determine directly from experiments and were determined through an inverse modeling procedure. Verification experiments were carried out by varying different environmental parameters and making comparisons with the simulated results using the corresponding parameters.<p> The experimental results showed that the proposed procedure is able to predict the amount of free chlorides in concrete, including predictions of chloride binding as a function of pH, temperature, chloride sources, and the presence of other ions such as carbonate. The proposed model was also used to clarify some unresolved issues such as the effect of chloride sources on binding and the effect of pH on the release of bound chlorides in the presence of carbonation.

The crystal structure of ATP-dependent phosphoenolpyruvate carboxykinase (ATP-oxaloacetate carboxy-lyase, (transphosphorylating), E.C. 4.1.1.49; PCK) from Escherichia coli K12 has been determined using a combination of multiple isomorphous replacement and density modification, and refined to a R-index of 0.202 (R-free = 0.244) at 1.9 A resolution. PCK catalyses the decarboxylation and ATP-dependent phosphorylation of oxaloacetate to form phosphoenolpyruvate, the first committed step of gluconeogenesis in E. coli. Each PCK molecule consists of a 275 residue N-terminal and 265 residue C-terminal ar mononucleotide-binding domain, with the active site located within a cleft between the two domains. PCK is an open-faced, mixed $\alpha/\beta$ protein with a unique overall tertiary structure. The putative phosphate-binding region of the ATP-binding site adopts the P-loop motif common to many ATP- and GTP-binding proteins. However, the â-sheet topology of the mononucleotide-binding fold of PCK differs from all other families within the P-loop containing nucleoside triphosphate hydrolase superfamily, suggesting PCK represents the first member of a new family of such proteins. The mononucleotide-binding domain also differs in structure from the classical mononucelotide-binding fold (CMBF), common to adenylate kinase, RecA, p21$\sp{{Ha}-ras}$, and elongation factor-Tu. Several highly-conserved amino acid residues among the ATP-dependent PCK family, including R65, Y207, K212, K213, H232, K254, T255, D269, K288 and R333 appear to make up the active site of the enzyme. A cysteine residue, C233, is located near the active site, and in the E. coli enzyme this residue is buried and is probably not involved in substrate binding or catalysis. Previous chemical modification studies, on several ATP- and GTP-dependent PCKs, have been assessed in view of these structural results. A mechanism of catalysis based on these and additional results is proposed. The structure of E. coli PCK complexed with the calcium-analogue Tb$\sp{3+}$ has been refined to an R-index of 0.205 (R-free = 0.259) at 2.5 A. Two binding sites for Tb$\sp{3+}$ have been determined, one within the active site coordinating to the side chains of K213, H232, and D269, and the other within the C-terminal domain, coordinating to the side chains of E508 and E511. No large structural movements are observed in PCK as a result of Tb$\sp{3+}$ binding, even though Ca$\sp{2+}$ is a known activator.

The chloride-induced corrosion of reinforcing steel in concrete structures has become a widespread durability problem throughout the world. When concrete structures come in contact with chloride sources, the chloride ions will diffuse through the body of the concrete and ultimately reach the steel. Not all of the chloride ions which penetrate the concrete remain free in the pore solution. Some of the ions become bound to the hydration products in a chemical reaction to form calcium chloroaluminate hydrate (Friedel' salt). It is also well known that only the portion of the chloride ions that remains free is responsible for causing damage to the concrete structures by corroding steel rebar. Thus, the chloride binding capacity of the cementitious matrix plays a major role in controlling chlorides ingress and, consequently, the corrosion of steel reinforcement in concrete. The chloride binding capacity is affected by cement composition, environmental factors, and by the source of the chlorides ( vs. ). To quantify the durability of new and existing structures, a clear understanding of the mechanisms of chloride penetration into the concrete cover is required.<p> Currently, most of the models available in the published literature for calculating free chloride ions in concrete use Ficks law for chloride transport and chloride binding isotherms to account for bound chlorides. Binding isotherms are cement and environment specific. Thus, the existing models cannot be used for all types of cement and variable general environmental exposure conditions such as temperatures, pH levels, and chloride sources. A general mechanistic approach that can overcome those limitations is proposed in this thesis based on the concepts of ion-exchange theory for an accurate determination of chloride ingress in concrete under variable environmental conditions.<p> Some of the model input parameters, such as exchange capacity and the equilibrium constant for the exchange reaction, were not easy to determine directly from experiments and were determined through an inverse modeling procedure. Verification experiments were carried out by varying different environmental parameters and making comparisons with the simulated results using the corresponding parameters.<p> The experimental results showed that the proposed procedure is able to predict the amount of free chlorides in concrete, including predictions of chloride binding as a function of pH, temperature, chloride sources, and the presence of other ions such as carbonate. The proposed model was also used to clarify some unresolved issues such as the effect of chloride sources on binding and the effect of pH on the release of bound chlorides in the presence of carbonation.

<p>Translesion synthesis (TLS) is a specialized type of DNA repair for bypassing DNA damage at the stalled replication fork. Because the TLS polymerases (mainly from the Y-family of polymerases) are characterized by more open active sites in order to accommodate the lesions, they are inherently more mutagenic than the replicative polymerases. Although essential for cell survival and in tolerating DNA damage, the access of the TLS polymerases to the replication fork must be tightly controlled. This regulation occurs in part through the ubiquitination state of the processivity factor PCNA. Damage-induced monoubiquitination of PCNA serves in part as the regulatory switch between replicative and translesion polymerases. Highly conserved ubiquitin-binding domains, the ubiquitin-binding zinc finger (UBZ) domain and the ubiquitin-binding motif (UBM), within the C-termini of the Y-family polymerases provide for an increased affinity of the polymerases to PCNA after damage to promote TLS. In order to determine the molecular basis for ubiquitin recognition by the TLS polymerases, we solved the solution structures of the human pol &#951; UBZ domain, the human pol &#953; UBM2 domain, and the human pol &#953; UBM2-ubiquitin complex. </p><p>The UBZ domain adopts a classical C2H2 zinc finger structure characterized by a &#946;&#946;&#945; fold, similar to the DNA-binding zinc finger proteins. Nuclear magnetic resonance titration mapped the binding interfaces between UBZ and ubiquitin to the &#945;-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single &#945;-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol &#951; UBZ domain represents a novel member of the C2H2 zinc finger family that interacts with ubiquitin to regulate translesion synthesis. </p><p>In contrast to the human UBZ domain, the yeast UBZ domain lacks one of the conserved cysteines necessary for zinc coordination, leading many to propose that it is a "zincless" zinc finger. We used biophysical methods to characterize the UBZ domains from human and yeast pol &#951; and to highlight differences between their structures and modes of ubiquitin binding. Like the human UBZ domain, the yeast UBZ domain binds zinc, which contributes to its secondary structure formation. In contrast to the human UBZ domain, the yeast UBZ domain binds to ubiquitin in a zinc-independent manner. Correspondingly, mutations in the zinc-coordinating residues of the yeast UBZ domain do not impair the polymerase's response to DNA damage.</p><p>We also investigated the structural elements and mechanism of ubiquitin recognition of the ubiquitin-binding motif (UBM) found in pol &#953; and Rev1. The solution structures of the C-terminal UBM of human pol &#953; and its complex with ubiquitin were solved. The UBM is a novel ubiquitin-binding domain that binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A of ubiquitin abolishes UBM binding. Human pol &#953; contains two functional UBMs, both of which contribute to replication foci formation. In contrast, only the second UBM of <italic>Saccharomyces cerevisiae</italic> Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations impairing the UBM-ubiquitin interaction also disrupt foci formation of pol &#953; and the DNA damage response of Rev1 <italic>in vivo</italic>, showing the significant role for the UBM in regulating TLS.</p><p>The structures of the UBZ domain and the UBM and their recognition of ubiquitin are different and distinct from other ubiquitin-binding domains. Their highly specific and unique associations with ubiquitin are critical for TLS regulation and further add to the diverse base of ubiquitin-binding domains and their role in mediating cellular functions.</p> / Dissertation

We synthesize polyamide compounds containing bithiophene functional group and lexitropsin analogs and study their biological activity.

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No description available.

Abstract Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Piscinodavirus (Betanodavirus) that causes vacuolating encephalopathy and retinopathy or viral nervous necrosis. MGNNV propagated in SSN-1 cells that were derived from fry tissue of striped snakehead fish, Channa striatus. SSN-1 was highly permissive to MGNNV infection and production, in which the TCID50 per ml increased from the magnitude of 10 4 to 10 8 after 5 passages. The thermal stability analysis unveiled that the optimal activity of MGNNV can be resumed at the temperature from frozen to less than 25 ¢J. Oreochromis mossambica was highly susceptible to MGNNV only when injecting the virus into the vitreous body of fish eye, via which the virus could propagate in the fish brain. Employing the technique of immobilizing virus on itrocellulose, several cellular MGNNV-binding proteins were detected. Two MGNNV-binding proteins of 100 and 56 kDa were found in SSN-1 cell lysate; four proteins of 60, 43, 36, and 30 kDa in rotifer lysate; four proteins of 62, 50, 20 and 25 kDa on the membrane of grouper fertilized eggs. In different organ lysates of Epinephelus malabaricus, two MGNNV-binding proteins of 80 and 43 kDa were found in brain; four MGNNV-binding proteins of 60, 45, 43 and 23 kDa were found in eye; two MGNNV-binding proteins of 80, 18 kDa were found in liver; four MGNNV-binding proteins of 60, 45, 43 and 30 kDa were found in pancreas; two MGNNV-binding proteins of 45 and 24 kDa were found in spleen; one MGNNV-binding proteins of 80 kDa were found in kidney; six MGNNV-binding proteins of 100, 95, 80, 48, 45 and 30 kDa were found in ovary.

The present study cloned successfully two lipid-metabolism genes, lipoprotein lipase (LPL) and fatty acid binding protein (FABPs) from cobia and studied the mRNA expressions of the two genes and their upstream gene PPARs when the cobia were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were supplemented by fish oil (FO, rich in n-3 HUFA), perilla oil (PE, rich in 18:2 n-6), safflower oil (SA, rich in 18:2 n-6), olive oil (OL, rich in 18:1 n-9) or palm oil (PA, rich in 16:0). The whole sequences of LPL, liver-FABP (L-FABP) and muscle-FABP (M-FABP) encode 520, 126 and 133 amino acids, respectively. RT-PCR and real time PCR analyses based on these gene sequences show that the mRNA expressions of L-FABP and M-FABP in the tissue of the cobia were diet-specific. The mRNA expression of LPL, on the other hand, did not respond to the treatments, except in visceral fat depot. Linear regression analysis shows that the mRNA expression of LPL in the liver and muscle was positively (P<0.05) related to dietary fatty acids and ther concentration, but that in the visceral fat depot was negatively related. The mRNA expression of FABPs was also positively correlated with dietary fatty acid levels. Among all fatty acids, the levels of C14:0, C20:1 n-9, EPA and DHA were positively correlated with the mRNA expression of PPAR£^and also with FABPs mRNA expression in the visceral fat depot and LPL mRNA expression in the muscle. Thus, LPL, L-FABP and M-FABP mRNA expression of the cobia were highly influenced by the kind and amount of dietary fatty acids. The role of PPARs was not clearly demonstrated.