Although a rare disease, light chain (LC) amyloidosis (AL) is the most common systemic amyloidosis in developed countries. It is caused by an overproduction of immunoglobulin LC proteins in bone marrow plasma cells. In AL amyloidosis, LCs that are prone to misfolding and insolubility will aggregate, form fibrils, and deposit themselves in various tissues, thereby causing organ dysfunction. The most fatal manifestation of AL amyloidosis is associated with cardiac involvement, defined by the presence of extracellular AL amyloid deposits within the heart. Cardiac amyloid infiltration typically leads to diastolic dysfunction followed by heart failure and has a median survival of approximately 6 months from the time of diagnosis if untreated. Clinical observation suggests that a reduction in circulating LCs results in an improvement in heart failure symptoms despite minimal changes in amyloid deposition. This has led to the concept that LCs themselves are cytotoxic to cardiomyocytes. Recent studies indicate that AL LCs induce oxidative stress, cellular dysfunction, and apoptosis (programmed cell death) in cardiomyocytes via a p38α mitogen-activated protein kinase (MAPK) mechanism. They may therefore be a target for amyloidosis therapy. By understanding how LCs cause cardiac dysfunction, we can target this process with therapies and utilize downstream measures of LC activity as diagnostic and prognostic tools. The objective of this study was to determine the role of autophagy in AL amyloidosis. Autophagy is the intracellular process of degrading aging or dysfunctional cellular components. Autophagy can be beneficial by preventing proteotoxicity and providing nutrients, amino acids, and other necessities during times of cellular stress. On the other hand, increased autophagy, like apoptosis, may mediate cellular death depending on the type of stimulus and its duration. Autophagy is induced by a variety of stimuli, including oxidative stress. AL has been demonstrated to increase reactive oxygen species (ROS), and it is unknown if autophagy mediates cardiomyocyte dysfunction in AL cardiac amyloidosis. We thus sought to determine if it is a factor in amyloid cardiotoxicity. We explored the ERK1/2, p38, and JNK MAPK pathways in particular, since MAPK signaling cascades regulate several transcription factors involved in the cell cycle and p38α has been implicated in ROS-induced cardiac AL amyloidosis. Adult rat ventricular myocytes (ARVM) were harvested from healthy adult male rats and exposed to a variety of experimental conditions in vitro. ARVM were treated with vehicle control, human LC obtained from a patient without cardiac involvement, a positive control (aldosterone), and human AL light chains obtained from a patient with AL cardiac amyloidosis in the presence or absence of UO126, SB203580, or SP600125 (specific inhibitors of ERK1/2, p38, and JNK, respectively). The resulting protein expression levels of autophagy indicators LC3II and ATG4B in cardiomyocytes were analyzed by Western blotting. The ratio of phosphorylated to total ERK1/2 protein expression was also explored. We found that AL light chains did not contribute to autophagy via the ERK1/2, p38, or JNK pathways. In contrast to our previous unpublished findings, the protein levels of autophagy indicators in AL-treated ARVM did not differ from vehicle control levels, suggesting that AL did not activate autophagy. However non-cardiomyopathic light chains (LC) did increase LC3II expression in ARVM, despite their human source exhibiting no clinical indications of cardiac involvement. This implies that autophagy induced by non-cardiomyopathic LCs may be beneficial and protect against the development of the cardiotoxicity seen in AL cardiac amyloidosis. Further studies are necessary to understand the effect of autophagy in the heart and its role in cardiac amyloidosis. Continuing to explore the underlying mechanisms of AL light chain toxicity will contribute to the development of diagnostic, prognostic, and treatment strategies for AL amyloidosis.

No description available.

Reliable biomarkers of axonal damage are urgently needed in neurological diseases. Neurofilaments (Nf) are specific structural elements of neurons composed of at least three subunits: Nf light chain (NfL), Nf medium and Nf heavy chain (NfH). This PhD aimed to characterise NfL levels and their correlation with clinical features in patients with neurological diseases with a different rate of progression and following and under different treatment regimes. An important aim was also to develop a bioassay for NfL measurements in blood. Cerebrospinal fluid (CSF) NfL levels discriminated patients with a clinically isolated syndrome (CIS) (p=0.001) or multiple sclerosis (MS) (p=0.035) from healthy controls more efficiently, and was more sensitive to change after natalizumab therapy (p<0.0001) than CSF NfH (p=0.002). Further, CSF NfL levels decreased in fingolimodtreated MS patients (p=0.001), but not in those receiving placebo (p=0.433). Based on these findings, a sensitive method for the detection of NfL in serum was developed and validated. Patients with neurological diseases had higher serum NfL values than controls. In acute spinal cord injury (SCI), serum NfL levels correlated with injury severity and long-term motor outcome, and Minocycline treatment was associated with decreased NfL levels in complete SCI patients compared to placebo. Finally, I found that serum NfL levels were higher in CIS patients than in healthy controls but did not predict conversion to clinically definite MS (CDMS). Independent predictors of CDMS were instead oligoclonal bands, number of T2 lesions and age at CIS. Lower 25-OHvitamin D levels were associated with CDMS in univariate analysis, but this was attenuated in the multivariate model. In conclusion, NfL proved to be an analytically stable protein which is an important prerequisite for biomarkers. The role of NfL quantification as a surrogate measure of neuroaxonal damage is corroborated by my findings and further supports the usefulness of NfL as a putative biomarker of axonal damage in various neurological diseases.

B lymphopoiesis in aged mice is characterized by reduced B cell precursors and an altered antibody repertoire. Aged mice maintain an ordinarily minor pool of early c-kit+ pre-B cells, indicative of poor preBCR expression, even as preBCR competent early pre-B cells are significantly reduced. Therefore, in aged mice, preBCR-mediated B2 B lymphopoiesis is significantly diminished; likely as a consequence of poor surrogate light chain expression. Notably, the remnant B1 B cell lineage present in adult bone marrow is retained in aged mice as evidenced by normal numbers (~0.3%) of Lin-CD19+B220low/- B1 B cell precursors. Of interest, B1 progenitors express substantially less lambda 5 surrogate light chain protein than do B2 pro-B cells and the surrogate light chain levels are further reduced in aged mice. B cells derived from putatively preBCR-deficient precursors, either B2 c-kit+ B cell precursors or B1 B cell progenitors, from either young or aged mice, generate new B cells in vitro that are biased to larger size, higher levels of CD43/S7, and decreased kappa light chain expression. Notably, immature B cells in aged bone marrow exhibit a similar phenotype in vivo, consistent with the changes seen in B cell precursor subpopulations. In aged mice, the B2 pathway is partially blocked with limited preBCR expression and signaling; however, continued B cell development via preBCR-deficient pathways, including B1 pathways, is observed. Increased generation of new B cells by these alternative pathways may contribute to altered phenotype, repertoire, and function in senescence.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

Increased exposure to ambient particulate matter (PM) is associated with elevated morbidity and mortality in patients with cardiopulmonary diseases and cancer. We and others have shown that PM induces lung microvascular barrier dysfunction which potentially enhances the systemic toxicity of PM. However, the mechanisms by which PM disrupts vascular endothelial integrity remain incompletely explored. We hypothesize that PM induces endothelial cell (EC) cytoskeleton rearrangement via Rho GTPase-dependent pathways to facilitate vascular hyperpermeability. Fine PM induced time-dependent activation of cytoskeletal machinery with increases in myosin light chain (MLC) phosphorylation and EC barrier disruption measured by transendothelial electrical resistance (TER), events attenuated by the Rho-dependent kinase (ROCK) inhibitor Y-27632 or the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC). Both Y-27632 and NAC prevented PM-induced stress fiber formation and phospho-MLC accumulation in human lung ECs. PM promotes rapid accumulation of Rho-GTP. This event is attenuated by NAC or knockdown of RhoA (siRNA). Consistent with ROCK activation, PM induced phosphorylation of myosin light chain phosphatase (MYPT) at Thr850, a post-translational modification known to inhibit phosphatase activity. Furthermore, PM activates the guanine nucleotide exchange factor (GEF) for Rho, p115, with p115 translocation to the cell periphery, in a ROS-dependent manner. Together these results demonstrate that fine PM induces EC cytoskeleton rearrangement via Rho-dependent pathways that are dependent upon the generation of oxidative stress. As the disruption of vascular integrity further contributes to cardiopulmonary physiologic derangements, these findings provide pharmacologic targets for prevention of PM-induced cardiopulmonary toxicity.

No description available.

No / L-type voltage gated calcium channels play essential role in contraction of various skeletal and vascular smooth muscles, thereby plays important role in regulating blood pressure. Dihydropyridine receptors have been targeted for development of newer antihypertensive agents, one of the structurally analogs nucleus dihydropyrimidines have been reported earlier by us as a potential agent toward development of calcium channel modulator. A pre-synthetic QSAR was run and on the basis of structure activity relationship a series of twenty three molecules was synthesized and studied by myosin light chain kinase assay (MLCK), Angiotensin Converting Enzyme (ACE) colorimetric assay, non-invasive blood pressure (NIBP) and invasive blood pressure (IBP) methods. Molecules with significant efficacy were studied for their single crystal X-ray diffraction, molecular docking, molecular dynamics and post-synthetic QSAR. The NIBP and IBP methods screened molecules with better percentage inhibition versus time compared to standard drug Nifedipine. The lead compound ethyl 2-methyl-4-(3-nitrophenyl)-4H-pyrimido [2,1-b] [1,3] benzothiazole-3-carboxylate (26) presented a triclinic structure with polymeric chain packing in lattice. 26 exhibited IC50 on MLCK assay of 2.1+/-1.7 muM with selectivity of L-type calcium channels and comparative to Nifedipine. It offered satisfactory physicochemical properties with partition coefficient of (ClogP) 4.64. Its pharmacokinetic profile is also good with Cmax at 0.40 mug/ml by oral route with Tmax reaching in 0.5 h which means in 30 min. 26 also exhibits superior t1/2 of 5.4 h and oral bioavailability of (F) 56.75% with an AUC0-infinity of 0.84 mug h/ml. Molecular docking studies indicates toward the interaction of lead compound via hydrogen bonds with Lys144, Glu181 and Asp183, it forms the Van der Walls interactions with Ser18, Asp20, Asn187, Pro185, Glu180, Glu181 and Arg10 with Glide score and Glide energy to be -3.602 and -47.098, respectively. Post-synthetic QSAR of newly synthesized molecules indicates toward improvement with respect to steric descriptor which contributed negatively in former series.

No description available.