Search

Objective: The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics., Methods: A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics., Results: We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC₂₀) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC₂₀ after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways., Conclusions: These data provide the first evidence that SP-mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation., (© 2010 John Wiley & Sons Ltd.)

The extracellular matrix (ECM) protein-integrin-cytoskeleton axis plays a central role as a mechanotransducing protein assemblage in many cell types. However, how the process of mechanotransduction and the mechanically generated signals arising from this axis affect myofilament function in cardiac muscle are not completely understood. We hypothesize that ECM proteins can regulate cardiac function through integrin binding, and thereby alter the intracellular calcium concentration ([Ca(2+)](i)) and/or modulate myofilament activation processes. Force measurements made in mouse papillary muscle demonstrated that in the presence of the soluble form of the ECM protein, fibronectin (FN), active force was increased significantly by 40% at 1 Hz, 54% at 2 Hz, 35% at 5 Hz and 16% at 9 Hz stimulation frequencies. Furthermore, increased active force in the presence of FN was associated with 12-33% increase in [Ca(2+)](i) and 20-50% increase in active force per unit Ca(2+). A function blocking antibody for α5 integrin prevented the effects of the FN on the changes in force and [Ca(2+)](i), whereas a function blocking α3 integrin antibody did not reverse the effects of FN. The effects of FN were reversed by an L-type Ca(2+) channel blocker, verapamil or PKA inhibitor. Freshly isolated cardiomyocytes exhibited a 39% increase in contraction force and a 36% increase in L-type Ca(2+) current in the presence of FN. Fibers treated with FN showed a significant increase in the phosphorylation of phospholamban; however, the phosphorylation of troponin I was unchanged. These results demonstrate that FN acts via α5β1 integrin to increase force production in myocardium and that this effect is partly mediated by increases in [Ca(2+)](i) and Ca(2+) sensitivity, PKA activation and phosphorylation of phospholamban., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Fast anionic oxy-Cope rearrangements of 1,5-hexadiyn-3,4-olates can be incorporated into cascade transformations which rapidly assemble densely functionalized cyclobutenes or cyclopentenones via a common bis-allenic intermediate. The competition between fragmentation, 4π-electrocyclic closure, and aldol condensation can be efficiently controlled by the nature of the acetylenic substituents. The rearrangement of bis-alkynes with two hydroxyl substituents opens a conceptually interesting entry in the chemistry of ε-dicarbonyl compounds and suggests a new approach to analogues of rocaglamide/aglafolin.

Hydride transfer from dihydronicotinamide adenine dinucleotide (NADH) analogues to manganese(V)-oxo corroles proceeds via proton-coupled electron transfer, followed by rapid electron transfer. The redox potentials (E(red)) of manganese(V)-oxo corroles exhibit a good correlation with their reactivity in hydride-transfer reactions.

Metabolic syndrome is defined by a cluster of different metabolic risk factors that include overall and central obesity, elevated fasting glucose levels, dyslipidemia, hypertension, and intimal atherogenesis. Metabolic syndrome leads to increased risk for the development of type 2 diabetes and cardiovascular disease (e.g., heart disease and stroke). The exacerbated progression of metabolic syndrome to cardiovascular disease has lead to intense study of the physiological ramifications of metabolic syndrome on the blood vasculature. These studies have particularly focused on the signaling and architectural alterations that manifest in hypertension and atherosclerosis. However, despite the overlap of metabolic syndrome pathology with lymphatic function, tangent effects on the lymphatic system have not been extensively documented. In this review, we discuss the current status of metabolic syndrome and provide evidence for, and the remaining challenges in studying, the connections among the lymphatic system, lipid transport, obesity, insulin resistance, and general inflammation., (© 2010 New York Academy of Sciences.)

The lymphatic system is composed of a dense network of lymphatic vessels, which are critical components of physiological interstitial fluid transport. These vessels possess intrinsic contractile properties providing the driving force for the fluid to be drained away from the tissues and propelled, as lymph, back into the bloodstream. Lymphatic pumping is also important to carry immune cells, bacteria, macromolecules, viruses and their products to and through lymph nodes, the other component of the lymphatic system, to initiate the adaptive immune response. In addition, among the many circulating mediators known to modulate lymphatic contractile activity and thus lymph flow, mediators of inflammation have potent excitatory or inhibitory actions. The involvement of lymphatic vessels in edema resolution, immune cell trafficking and their sensitivity to inflammatory mediators make them pivotal players of the inflammation process. The ability of lymphatic vessels to generate and regulate lymph flow is provided by the lymphatic muscle present in the vessels' wall. Although molecular studies investigating the mechanisms of lymphatic vessel contraction are still very limited, recent findings suggest that lymphatic pumping requires complicated muscle activities that have similarities to those seen in both the heart (striated muscle) and blood vessels (smooth muscle). This review article focuses on presenting and discussing the mechanisms that regulate lymphatic vessel contraction under normal and pathophysiological states, specifically pertaining to inflammatory conditions., (Copyright © 2009. Published by Elsevier Ireland Ltd.)

Hundreds of proteins are inserted post-translationally into the endoplasmic reticulum (ER) membrane by a single carboxy-terminal transmembrane domain (TMD). During targeting through the cytosol, the hydrophobic TMD of these tail-anchored (TA) proteins requires constant chaperoning to prevent aggregation or inappropriate interactions. A central component of this targeting system is TRC40, a conserved cytosolic factor that recognizes the TMD of TA proteins and delivers them to the ER for insertion. The mechanism that permits TRC40 to find and capture its TA protein cargos effectively in a highly crowded cytosol is unknown. Here we identify a conserved three-protein complex composed of Bat3, TRC35 and Ubl4A that facilitates TA protein capture by TRC40. This Bat3 complex is recruited to ribosomes synthesizing membrane proteins, interacts with the TMDs of newly released TA proteins, and transfers them to TRC40 for targeting. Depletion of the Bat3 complex allows non-TRC40 factors to compete for TA proteins, explaining their mislocalization in the analogous yeast deletion strains. Thus, the Bat3 complex acts as a TMD-selective chaperone that effectively channels TA proteins to the TRC40 insertion pathway.

The morphological and genetic diversity of cyanobacteria associated with cycads was examined using PCR amplification techniques and 16S rRNA gene sequence analysis. Eighteen symbiotic cyanobacteria were isolated from different cycad species. One of the symbiotic isolates was a species of Calothrix, a genus not previously reported to form symbioses with Cycadaceae family, and the remainder were Nostoc spp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the repetitive sequences. Based on fingerprint patterns and phenograms, it was revealed that cyanobacterial symbionts exhibit important genetic diversity among host plants, both within and between cycad populations. A phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that most of the symbiotic cyanobacterial isolates fell into well-separated clades., (((c) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

Integrins link the extracellular matrix (ECM) with the intracellular cytoskeleton and other cell adhesion-associated signaling proteins to function as mechanotransducers. However, direct quantitative measurements of the cardiomyocyte mechanical state and its relationship to the interactions between specific ECM proteins and integrins are lacking. The purpose of this study was to characterize the interactions between the ECM protein fibronectin (FN) and integrins in cardiomyocytes and to test the hypothesis that these interactions would vary during contraction and relaxation states in cardiomyocytes. Using atomic force microscopy, we quantified the unbinding force (adhesion force) and adhesion probability between integrins and FN and correlated these measurements with the contractile state as indexed by cell stiffness on freshly isolated mouse cardiomyocytes. Experiments were performed in normal physiological (control), high-K(+) (tonically contracted), or low-Ca(2+) (fully relaxed) solutions. Under control conditions, the initial peak of adhesion force between FN and myocyte alpha(3)beta(1)- and/or alpha(5)beta(1)-integrins was 39.6 +/- 1.3 pN. The binding specificity between FN and alpha(3)beta(1)- and alpha(5)beta(1)-integrins was verified by using monoclonal antibodies against alpha(3)-, alpha(5)-, alpha(3) + alpha(5)-, or beta(1)-integrin subunits, which inhibited binding by 48%, 65%, 70%, or 75%, respectively. Cytochalasin D or 2,3-butanedione monoxime (BDM), to disrupt the actin cytoskeleton or block myofilament function, respectively, significantly decreased the cell stiffness; however, the adhesion force and binding probability were not altered. Tonic contraction with high-K(+) solution increased total cell adhesion (1.2-fold) and cell stiffness (27.5-fold) compared with fully relaxed cells with low-Ca(2+) solution. However, it could be partially prevented by high-K(+) bath solution containing BDM, which suppresses contraction by inhibiting the actin-myosin interactions. Thus, our results demonstrate that integrin binding to FN is modulated by the contractile state of cardiac myocytes.

A convenient, fast and high yielding method for the preparation of 7-methoxyisoflavanone and 2,3-diarylchromanones has been developed by the condensation of benzyl-2-hydroxy-4-alkoxyphenylketone with arylaldehyde/paraformaldehyde in presence of diethylamine, assisted by microwave activation. All the synthesized compounds were screened for anticancer as well as antioxidant activities. Among the nine compounds, 7-methoxyisoflavanone 7 and diarylchromanone 6c shows potential anticancer activity and diarylchromanone 6b has potential antioxidant activity. Compound 6h possesses anticancer and antioxidant activity at the same concentration., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

Mediastinal diffuse large B-cell lymphoma (Med-DLBCL) is a subtype of DLBCL that has morphologic and clinical similarities and phenotypic overlaps with classical Hodgkin lymphoma (CHL) involving the mediastinum. CD23 is a marker that has been previously reported in Med-DLBCI and is proposed in the differential diagnosis of M-DLBCL and CHL. A panel of immunostains, including CD45, CD20, CD3, CD30, CD15, CD21, and CD23 as well as Eber was performed on Med-DLBCL and 20CHL. 23/27 Med-DLBCL (85%) were positive for CD23 (membranous) CD30 was negative in 6 and positive in 21 cases. 18 CHL cases were negative for CD23 and only 2 showed rare scattered Reed-Sternberg cells with weak cytoplasmic CD23 staining. CD23 showed a sensitivity of 85% and positive predictive value of 92%. In conclusion CD23 is a useful marker in distinguishing Med-DLBCL and CHL in mediastinal biopsies and may be helpful as an adjunct to histomorphology and other markers in the diagnosis and appropriate clinical management of these lesions.

The aerobic reaction of the Schiff-base ligand N-(benzimidazol-2-yl)salicylaldimine (Hbisi) with iron(II) perchlorate in methanol leads to the formation of the remarkable coordination compound [Fe(4)(mu(4)-O)(mu-MeO)(4)(bisi)(4)](ClO(4))(2) x 4 MeOH (1), whose single-crystal X-ray structure reveals the presence of a discrete Fe(III)(4)(mu(4)-O) core. Magnetic and Mossbauer studies both show that the exchange interaction within the square tetranuclear iron(III) unit is dominated by the central bridging mu(4)-oxido ligand, the involvement of the mu-methoxido bridges being negligible.

In eukaryotic cells, roughly one-fourth of all mRNAs code for secretory and membrane proteins. This class of proteins must first be segregated to the endoplasmic reticulum, where they are either translocated into the lumen or inserted into the lipid bilayer. The study of these processes has long relied on their successful reconstitution in cell-free systems. The high manipulability of such in vitro systems has allowed the identification of key machinery, elucidation of their functional roles in translocation, and dissection of their mechanisms of action. Here, we provide the basic methodology for (i) setting up robust mammalian-based in vitro translation and translocation systems, (ii) assays for protein translocation, insertion, and topology, and (iii) methods to solubilize, fractionate, and reconstitute ER membranes. Variations of these methods should be applicable not only to forward protein translocation systems but also for dissecting other poorly understood membrane-associated processes such as retrotranslocation.

Surra is an animal pathogenic protozoan infection, caused by Trypanosoma evansi, that develops into a fatal wasting disease. Control measures rely on diagnosis and treatment. However, with the continuous emergence of drug resistance, this tactic is failing, and the pressing need for new chemotherapeutic agents is becoming critical. With the introduction of novel aromatic diamidines, a new category of antitrypanosomal drugs was discovered. Nevertheless, their efficacy within a T. evansi-infected mouse model was not known. In total, 30 compounds previously selected based on their in vitro activity were tested in a T. evansi mouse model of infection. Six of the compounds were capable of curing T. evansi-infected mice at drug doses as low as 0.5 and 0.25 mg/kg of body weight administered for 4 consecutive days, and they were more effective than the standard drugs suramin, diminazene, and quinapyramine. After all selection criteria were applied, three diamidine compounds (DB 75, DB 867, and DB 1192) qualified as lead compounds and were considered to have the potential to act as preclinical candidates against T. evansi infection.

Depending on the reaction conditions and the nature of substituents at the triple bond, anionic cyclizations of hydrazides of o-acetylenyl benzoic acids can be selectively directed along three alternative paths, each of which provides efficient access to a different class of nitrogen heterocycles. The competition between 5-exo and 6-endo cyclizations of the "internal" nitrogen nucleophile is controlled by the nature of alkyne substituents under the kinetic control conditions. In the presence of KOH, the initially formed 5-exo products undergo a new rearrangement that involves a ring-opening followed by recyclization to the formal 6-exo-products and rendered irreversible by a prototropic isomerization. DFT computations provide insight into the nature of factors controlling relative rates of 5-exo, 6-endo, and 6-exo cyclization paths, ascertain the feasibility of direct 6-exo closure and relative stability for the anionic precursor for this process, provide, for the first time, the benchmark data for several classes of anionic nitrogen cyclizations, and dissect stereoelectronic effects controlling relative stability of cyclic anionic intermediates and influencing reaction stereoselectivity. We show that the stability gain due transformation of a weak pi-bond into a stronger sigma-bond (the usual driving force for the cyclizations of alkynes) is offset in this case by the transformation of a stable nitrogen anion into an inherently less stable carbanionic center. As a result, the cyclizations are much more sensitive to external conditions and substituents than similar cyclizations of neutral species. However, the exothermicity of such anionic cyclizations is increased dramatically upon prototropic isomerization of the initially formed carbanions into the more stable N-anions. Such tautomerizations are likely to play the key role in driving such cyclizations to completion but may also prevent future applications of such processes as the first step in domino cyclization processes.

Objective: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself., Methods: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells., Results: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels., Conclusions: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are 'tail-anchored' by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP.AlF(4)(-)-bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC(20)) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC(20) (pMLC(20)) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1-10 microM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cm H(2)O) and substance P (SP; 10(-7) M). Immunohistochemical and Western blot analyses of pMLC(20) were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC(20); 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC(20) phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent.

Large amplitude molecular switches have been developed using oxonium ions as the novel switching mechanism. Macrocycles that contain a polyether ring that are preorganized and of optimum geometry such that strong, linear Low-Barrier Hydrogen Bonds (LBHB, 2.4 to 2.6 A in length) are formed between a protonated amide oxygen and a cyclic ether, that lend significant iminol character to the amide. Deprotonation yields a large conformational change between closed and open forms, mindful of a new hinged, latch-type mechanical proton switch. Numerous open and closed forms have been characterized by X-ray crystallography, and the intramolecular hydrogen bond that forms between the protonated amide oxygen and the cyclic polyether oxygen accounts for the stability of these new acids. The open form of the deprotonated adducts persist in solution as indicated by the magnitude of coupling constants and other Nuclear Overhauser Effect experiments. Different saturated and unsaturated solid acids have been characterized including products derived from acetonitrile, propionitrile, caprylonitrile, acrylonitrile and adiponitrile, and also by reaction with primary amides in the case of phenyl and norbornene derivatives. We have also demonstrated that metal cations can replace the proton in the switching mechanism, characteristic of nascent synthetic pores.

Ambient protein levels are affected by both synthesis and degradation. Synthesis of a protein is regulated by transcription and messenger RNA (mRNA) translation. Translation has emerged as an important site of regulation of protein expression during development and disease. It is under the control of distinct factors that regulate initiation, elongation and termination phases. Regulation of translation occurs via signaling reactions, guanosine diphosphate-guanosine triphosphate binding and by participation of non-coding RNA species such as microRNA. Recent work has revealed an important role for translation in hypertrophy, matrix protein synthesis, elaboration of growth factors in in vivo and in vitro models of diabetic nephropathy. Studies of translation dysregulation in diabetic nephropathy have enabled identification of novel therapeutic targets. Translation of mRNA is a fertile field for exploration in investigation of kidney disease.

This work deals with atmospheric corrosion to assess the degrading effects of air pollutants on ferrous and non-ferrous metals and alloys, which are mostly used as engineering materials. An exposure study was conducted in the Tuticorin port area located on the east coast of South India, in the Gulf of Mannar with Sri Lanka to the southeast. Common engineering materials, namely mild steel, galvanized iron, Zn, Al, Cu and Cu-Zn alloys (Cu-27Zn, Cu-30Zn and Cu-37Zn), were used in the investigation. The site was chosen where the metals are exposed to marine and industrial atmospheres. Seasonal 1 to 12 month corrosion losses of these metals and alloys were determined by a weight loss method. The weight losses showed strong corrosion of mild steel, galvanized iron, Cu and Zn and minor effect on Al and Cu-Zn alloys. Linear regression analysis was conducted to study the mechanism of corrosion. The composition of corrosion products formed on the metal surfaces was identified by x-ray diffraction and Fourier transform infrared spectroscopy.

Oligomeric o-aryleneethynylenes with three triple bonds undergo cascade radical transformations in reaction with a Bu 3SnH/AIBN system. These cascades involve three consecutive cycle closures with the formation of substituted benzo[ a]indeno[2,1- c]fluorene or benzo[1,2]fluoreno[4,3- b]silole derivatives. The success of this sequence depends on regioselectivity of the initial attack of the Bu 3Sn radical at the central triple bond of the o-aryleneethynylene moiety. The cascade is propagated through the sequence of 5-exo-dig and 6-exo-dig cyclizations which is followed by either a radical attack at the terminal Ar substituent or radical transposition which involves H-abstraction from the terminal TMS group and 5-endo-trig cyclization. Overall, the transformation has potential to be developed into an approach to a new type of graphite ribbons.

Despite being predicted to be stereoelectronically favorable by the Baldwin rules, efficient formation of a C-C bond through a 5-endo-dig radical cyclization remained unknown for more than 40 years. This work reports a remarkable increase in the efficiency of this process upon beta-Ts substitution, which led to the development of an expedient approach to densely functionalized cyclic 1,3-dienes. Good qualitative agreement between the increased efficiency and stereoselectivity for the 5-endo-dig cyclization of Ts-substituted vinyl radicals and the results of density functional theory analysis further confirms the utility of computational methods in the design of new radical processes. Although reactions of Br atoms generated through photochemical Ts-Br bond homolysis lead to the formation of cyclic dibromide side products, the yields of target bromosulfones in the photochemically induced reactions can be increased by recycling the dibromide byproduct into the target bromosulfones through a sequence of addition/elimination reactions at the exocyclic double bond. Discovery of a relatively efficient radical 5-endo-dig closure, accompanied by a C-C bond formation, provides further support to stereoelectronic considerations at the heart of the Baldwin rules and fills one of the last remaining gaps in the arsenal of radical cyclizations.

Substance P (SP) is a neuropeptide associated with sensory innervation of lymphoid tissue and a suspected modulator of lymphatic function in inflammation. Only a few studies have examined the effects of SP on lymphatic contraction, and it is not clear to what extent SP acts directly on the lymphatic muscle and/or endothelium or indirectly through changes in intraluminal filling pressure secondary to increases in capillary permeability/filtration. We tested the effects of SP on the spontaneous contractions of rat isolated mesenteric lymphatic vessels under isometric and isobaric conditions, hypothesizing that low concentrations would stimulate lymphatic pumping by enhancing lymphatic muscle contraction in a manner complementary to the effect of increased preload. Under isometric conditions, SP (10 nM) dramatically enhanced lymphatic chronotropy and inotropy. Unlike guinea pig lymphatics, SP actions were not blocked by cyclooxygenase or PLA(2) inhibition. In the absence of SP, ramp increases in isometric preload resulted in x approximately 1.6 increases in contraction amplitude (Amp) and x approximately 1.7 increases in frequency (Freq). SP increased Freq by x approximately 2.4, Amp by x approximately 1.9, and the Amp-Freq product (AFP) by x approximately 3.5. Under isobaric conditions, the pressure elevation from 0.5 to 10 cmH(2)O in the absence of SP decreased Amp by x approximately 0.6 and increased Freq by x approximately 1.8. SP caused a modest increase in Amp, a robust increase in Freq at all pressures, and shifted the AFP-pressure relationship upward and leftward. Therefore, SP has substantial positive inotropic and chronotropic effects on rat lymphatic muscle, improving pump efficiency independent of the effects of preload and broadening of the working range of the lymphatic pump.

Lymphatic muscle contraction is critical for the centripetal movement of lymph that regulates fluid balance, protein homeostasis, lipid absorption, and immune function. We have demonstrated that lymphatic muscle has both smooth and striated muscle contractile elements; however, the basic contractile properties of this tissue remain poorly defined. We hypothesized that contractile characteristics of lymphatic myofilaments would be different from vascular smooth muscle myofilaments. To test this hypothesis, -log[Ca(2+)] (pCa)-tension relationship was determined for alpha-toxin permeabilized mesenteric lymphatics, arteries, and veins. The Ca(2+) sensitivity (pCa(50)) of mesenteric lymphatics was significantly lower compared with arteries (6.16 +/- 0.05 vs. 6.44 +/- 0.02; P < 0.05), whereas there was no difference in pCa(50) between lymphatics and veins (6.16 +/- 0.05 vs. 6.00 +/- 0.10; not significant). The Hill coefficient for alpha-toxin-permeabilized lymphatics was not significantly different from arteries but was significantly greater than that of the veins (1.98 +/- 0.19 vs. 1.21 +/- 0.18; P < 0.05). In addition, the maximal tension and pCa(50) values were significantly greater in alpha-toxin-permeabilized lymphatics compared with beta-escin-permeabilized lymphatics (0.27 +/- 0.03 vs. 0.15 +/- 0.01 and 6.16 +/- 0.05 vs. 5.86 +/- 0.06 mN/mm, respectively; P < 0.05), whereas the Hill coefficient was significantly greater in beta-escin-permeabilized lymphatics. Western blot analyses revealed that CPI-17 levels were significantly decreased by about 50% in beta-escin-permeabilized lymphatics, compared with controls, whereas no change in the level of calmodulin was detected. Our data constitute the first description of the pCa-tension relationship in permeabilized lymphatic muscle. It suggests that differences in myofilament Ca(2+) sensitivity and cooperativity among lymphatic muscle and vascular smooth muscles contribute to the functional differences that exist between these tissues.

Introduction: Mucocele-like tumor was originally described in 1986 as a benign breast proliferation consisting of multiple dilated cysts lined by cytologically bland, flat to cuboidal cells. Subsequent reports described the coexistence of, including the morphologic inter-transitions between, mucocele-like tumor and a variety of other breast proliferations, including intraductal carcinoma, invasive carcinoma, atypical ductal hyperplasia, and hyperplasia of the usual type. The spectrum of breast alterations characterized by variably enlarged terminal-ductal lobular units lined by variably hyperplastic and variably atypical columnar cells has been the subject of significant discussion in the recent literature. In one scheme, these lesions may be classified into four groups, that is, columnar cell change with and without atypia and columnar cell hyperplasia with and without atypia. Morphologic and molecular observations suggest an association, perhaps in a nonobligate precursor role, between some columnar cell lesions and a variety of other neoplastic lesions., Case Presentation: We describe the case of a 43-year-old woman whose breast tumor contained areas diagnostic of mucocele-like tumor and columnar cell hyperplasia, with morphologic transitions in between., Conclusion: Our case represents the second broadly similar case that has been reported, and suggests a potential relationship between these two enigmatic lesions.

A series of mixed ligand ruthenium(II) complexes [Ru(Hdpa)2(diimine)](ClO4)2, 1-5 where Hdpa is 2,2'-dipyridylamine and diimine is 1,10-phenanthroline (phen) and a modified/extended 1,10-phenanthroline such as, 5,6-dimethyl-1,10-phenanthroline (5,6-dmp), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), 5-methyldipyrido[3,2-d:2',3'-f]quinoxaline (mdpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) have been isolated and characterized by analytical and spectral methods. The complex [Ru(Hdpa)2(phen)](PF6)2 1 has been structurally characterized and the coordination geometry around Ru(II) in it is described as distorted octahedral. 1H NMR spectral data reveal that 1-5 should have a C2 symmetry lying on the diimine plane due to the rapid flapping of the coordinated Hdpa ligands. The interaction of the complexes with calf thymus (CT) DNA has been explored by using absorption and emission spectral and viscometry and electrochemical techniques and the mode of DNA binding of the complexes has been proposed. The DNA binding affinity of the complexes decreases with decrease in number of planar aromatic rings in the co-ligand supporting the intercalation of the diimine co-ligands in between the DNA base pairs. Circular dichroic spectral studies reveal that the complexes 3-5 exhibit induced circular dichroism upon binding to CT DNA. Interestingly, upon interaction with CT DNA all the complexes show an increase in anodic current in the cyclic voltammograms suggesting that they are involved in electrocatalytic guanine oxidation. Interestingly, of all the complexes, only 5 alters the DNA superhelicity upon binding with supercoiled pBR322 DNA, which is consistent with its higher DNA binding affinity. Further, the cytotoxicities of the complexes against human cervical epidermoid carcinoma cell line (ME180) have been examined. Interestingly, 5 exhibits a cytotoxicity against ME180 higher than other complexes with potency approximately 8 times more than cisplatin for 24 h incubation but 4 times lower than cisplatin for 48 h incubation.

Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.

Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.

Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the oxidation of a specific cysteine residue to formylglycine, which is the catalytic residue required for sulfate ester hydrolysis. This post-translational modification occurs in the endoplasmic reticulum (ER), and is an essential step in the biogenesis of this enzyme family. A paralog of FGE (pFGE) also localizes to the ER. It shares many properties with FGE, but lacks formylglycine-generating activity. There is evidence that FGE and pFGE act in concert, possibly by forming complexes with sulfatases and one another. Here we show that human pFGE, but not FGE, is retained in the ER through its C-terminal tetrapeptide PGEL, a noncanonical variant of the classic KDEL ER-retention signal. Surprisingly, PGEL, although having two nonconsensus residues (PG), confers efficient ER retention when fused to a secretory protein. Inducible coexpression of pFGE at different levels in FGE-expressing cells did not significantly influence the kinetics of FGE secretion, suggesting that pFGE is not a retention factor for FGE in vivo. PGEL is accessible at the surface of the pFGE structure. It is found in 21 mammalian species with available pFGE sequences. Other species carry either canonical signals (eight mammals and 26 nonmammals) or different noncanonical variants (six mammals and six nonmammals). Among the latter, SGEL was tested and found to also confer ER retention. Although evolutionarily conserved for mammalian pFGE, the PGEL signal is found only in one further human protein entering the ER. Its consequences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved.

The hepatoprotective potential of earthworm extract (EE) (Lampito mauritii, Kinberg) was evaluated against paracetamol-induced liver injury in Wistar albino rat, in comparison with silymarin, the standard hepatoprotective drug. We observed a reduction in liver antioxidants, such as glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) and in serum total protein, and an increase in serum alkaline phosphatase (ALP), serum aspertate aminotranferase (AST), serum alanine aminotranferase (ALT), bilirubin and liver thiobarbituric acid reactive substances (TBARS) due to liver injury in the paracetamol-administered rats (2 g/kg). On the contrary, increased activities of liver GSH, SOD, GPx, CAT and serum total protein level, and decrease in the contents of serum ALP, AST, ALT, bilirubin and liver TBARS were observed in rats administered with different doses of EE (100, 200 and 300 mg/kg), which are similar to the activities of hepatoprotective drug silymarin (150 mg/kg). The mode of action of EE as evidenced by the above parameters may suggest that EE, on the one hand, prevents the formation of the reactive oxygen groups, or scavenges these groups, thereby preventing the damage on the hepatic cells, and, on the other hand, modulates the genes responsible for synthesis of antioxidant enzymes such as GPx, CAT and SOD in liver tissue and decreases the serum enzymatic activities such as ALP, AST and ALT.

A series of mixed ligand ruthenium(II) complexes [Ru(pdto)(diimine)](ClO4)2/(PF6)2 1-3 and [Ru(bbdo)(diimine)](ClO4), 4-6, where pdto is 1,8-bis(pyrid-2-yl)-3,6-dithiooctane, bbdo is 1,8-bis(benzimidazol-2-yl)-3,6-dithiooctane and diimine is 1,10-phenanthroline (phen), dipyrido-[3,2-d:2',3'-f]-quinoxaline (dpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz), have been isolated and characterised by analytical and spectral methods. The complexes [Ru(pdto)(phen)](PF6)2 la, [Ru(pdto)(dpq)(Cl](PF6) 2a, [Ru(bbdo)(phen)](PF6)2 4a and [Ru(bbdo)(dpq)](ClO4)2 5 have been structurally characterized and their coordination geometries around ruthenium(II) are described as distorted octahedral. In la, 4a and 5 the two thioether sulfur and two py/bzim nitrogen atoms of the tetradentate pdto/bbdo ligand are folded around Ru(II) to give predominantly a "cis-alpha" configuration. (I)H NMR spectral data of the complexes support this configuration in solution. In [Ru(pdto)(dpq)Cl](PF6) 2a with a distorted octahedral coordination geometry, one of the two py nitrogens of pdto is not coordinated. The DNA binding constants (Kb: 2, 2.00 +/- 0.02 x 10(4) M(-1), s = 1.0; 3, 3.00 +/- 0.01 x 10(6) M(-1), s = 1.3) determined by absorption spectral titrations of the complexes with CT DNA reveal that 3 interacts with DNA more tightly than 2 through partial intercalation of the extended planar ring of coordinated dppz with the DNA base stack. The DNA binding affinities of the complexes increase with increase in the number of planar aromatic rings in the co-ligand, and on replacing both the py moieties in pdto complexes (1-3) by bzim moieties to give bbdo complexes (4-6). Upon interaction with CT DNA the complexes 1, 2, 5 and 6 show a decrease in anodic current in the cyclic voltammograms. On the other hand, interestingly, 3 and 4 show an increase in anodic current suggesting their involvement in electrocatalytic guanine oxidation. Interestingly, of all the complexes, only 6 alters the superhelicity of DNA upon binding with supercoiled pBR322 DNA. The cytotoxicities of the dppz complexes 3 and 6, which avidly bind to DNA, have been examined by screening them against cell lines of different cancer origins. It is noteworthy that 6 exhibits selectivity with higher cytotoxicity against the melanoma cancer cell line (A375) than other cell lines, potency approximately twice that of cisplatin and toxicity to normal cells 3 and 90 times less than cisplatin and adriamycin respectively.

The lymphatic system plays critical roles in body fluid and macromolecular homeostasis, lipid absorption, immune function, and metastasis. To accomplish these tasks, the lymphatics must move lymph and its contents from the interstitial space through the lymph vessels and nodes and into the great veins. Contrary to popular belief, lymph does not passively "drain" down this pathway, because the net pressure gradients oppose flow. Instead, the lymphatics must act as both the conduits that direct and regulate lymph flow and the pumps that generate the lymph flow. Thus, to regulate lymph transport and function, both lymphatic pumping and flow resistance must be controlled. Both of these processes occur via regulation of lymphatic muscle contractions, which are classically thought to occur via the interaction of cell calcium with regulatory and contractile proteins. However, our knowledge of this regulation of lymphatic contractile function is far from complete. In this chapter we review our understanding of the important molecular mechanisms, the calcium regulation, and the contractile/regulatory proteins that control lymphatic contractions. A better understanding of these mechanisms could provide the basis for the development of better diagnostic and treatment modalities for lymphatic dysfunction. While progress has been made in our understanding of the molecular biology of lymphangiogenesis as a result of the development of potential lymphangiogenic therapeutic targets, there are currently no therapeutic agents that specifically modulate lymphatic pump function and lymph flow via lymphatic muscle. However, their development will not be possible until the molecular basis of lymphatic contractility is more fully understood.

Objective: To evaluate the age-related changes in active pumping in thoracic duct (TD) from 24-month-old Fisher-344 rats comparing with TD pumping in 9-month rats., Methods: Lymphatic diameters, contraction amplitude and frequency, ejection fraction, and fractional pump flow were determined in isolated TD preparations. Western blot analyses were performed to evaluate relative levels of eNOS and iNOS in 9- and 24-month-old TD., Results: Stretch-dependent regulation was altered in aged TD especially at higher levels of pressure: the negative inotropy, negative chronotropy and diminished minute pumping (2- to 3-fold decrease) were observed. Physiological NO/imposed-flow-dependent inhibition was completely abolished in aged TD, yet NO-synthase blockade by L-NAME (10(-4) M) increased pumping in a flow-independent manner. Western blot analyses indicated that the relative levels of eNOS were decreased approximately 7-fold in the 24-month-old TD when compared with 9-month-old TD; whereas iNOS levels were increased approximately 10-fold in 24-month-old TD., Conclusions: These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.

We present a method for stochastic fiber tract mapping from diffusion tensor MRI (DT-MRI) implemented on graphics hardware. From the simulated fibers we compute a connectivity map that gives an indication of the probability that two points in the dataset are connected by a neuronal fiber path. A Bayesian formulation of the fiber model is given and it is shown that the inversion method can be used to construct plausible connectivity. An implementation of this fiber model on the graphics processing unit (GPU) is presented. Since the fiber paths can be stochastically generated independently of one another, the algorithm is highly parallelizable. This allows us to exploit the data-parallel nature of the GPU fragment processors. We also present a framework for the connectivity computation on the GPU. Our implementation allows the user to interactively select regions of interest and observe the evolving connectivity results during computation. Results are presented from the stochastic generation of over 250,000 fiber steps per iteration at interactive frame rates on consumer-grade graphics hardware.

Follicular lymphomas are frequently associated with the t(14;18)(q32;q21). This translocation can be detected by karyotype, polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). In addition to the breakpoints currently used for diagnosis located in the major breakpoint region (MBR) and the minor cluster region (mcr), recent studies have reported the existence of other breakpoints (3' BCL2, 5'mcr, and icr). In this study, we examined the frequency of all five breakpoints in 236 cases of follicular lymphomas by real-time PCR analysis. The distribution of breakpoint sites consisted of MBR in 118 cases (50%), mcr in 11 (5%), icr in 32 (13%), 3' BCL2 in 13 (6%), and 5' mcr in three cases (1%). These findings illustrate significantly higher frequency of the icr breakpoint as compared with the more frequently studied mcr. Correlation of breakpoints with histology showed that MBR breakpoints occur more frequently in grade 2 lymphomas (P = 0.042). A majority of the PCR-negative cases (75%) contained an IGH/BCL2 translocation with FISH methods, suggesting the presence of other BCL2 breakpoints. Correlation of breakpoints with survival did not reveal significant differences. Diagnostic laboratories should consider expanding their PCR methods to include other BCL2 breakpoints and correlating with FISH methods when appropriate.

Human African trypanosomiasis is a devastating disease with only a few treatment options, including pentamidine. Diamidine compounds such as pentamidine, DB75, and DB820 are potent antitrypanosomal compounds. Previous investigations have shown that diamidines accumulate to high concentrations in trypanosomes. However, the mechanism of action of this class of compounds remains unknown. A long-hypothesized mechanism of action has been binding to DNA and interference with DNA-associated enzymes. The fluorescent diamidines, DB75 and DB820, have been shown to localize not only in the DNA-containing nucleus and kinetoplast of trypanosomes but also to the acidocalcisomes. Here we investigate two series of analogs of DB75 and DB820 with various levels of in vitro antitrypanosomal activity to determine whether any correlation exists between trypanosome accumulation, distribution, and in vitro activity. Despite wide ranges of in vitro antitrypanosomal activity, all of the compounds investigated accumulated to millimolar concentrations in trypanosomes over a period of 8 h. Interestingly, some of the less potent compounds accumulated to concentrations much higher than those of more potent compounds. All of the compounds were localized to the DNA-containing nucleus and/or kinetoplast, and many were also found in the acidocalcisomes. Accumulation in the nucleus and kinetoplast should be important to the mechanism of action of these compounds. The acidocalcisomes may also play a role in the mechanism of action of these compounds. This investigation suggests that the extent of accumulation alone is not responsible for killing trypanosomes and that organelle-specific accumulation may not predict in vitro activity.

Background: Primary lymphoma of the breast has been reported to have a high local and central nervous system recurrence (CNS) rate, suggesting the need for consolidation radiotherapy and CNS prophylaxis. A retrospective study was done to evaluate the institutional experience in this patient population., Methods: In all, 37 patients with lymphoma involving the breast at initial diagnosis and managed at Stanford University from 1981-2005 were included. Diagnostic tissue biopsies were obtained either from the breast mass or an involved lymph node. Treatment and response data, patterns of recurrence, and outcomes were reviewed., Results: Diffuse large B cell lymphoma (DLBCL) was the most common histologic subtype seen in 18 of 37 (49%) patients. Follicular and marginal zone subtypes were seen in 38%. Most patients presented with an incidental breast mass in stage I(E) or II(E). Four (11%) patients presented with bilateral breast involvement, with only 1 patient presenting with CNS disease. DLBCL patients received doxorubicin-based chemotherapy, with 70% receiving involved field radiotherapy and a single patient receiving intrathecal therapy. No recurrences occurred in the involved breast and a single parenchymal CNS recurrence was recorded. Among the DLBCL patients, the 5-year progression-free survival (PFS) was 61%, with a median follow-up of 3.8 years (range, 5 months to 19 years) and the 5-year overall survival (OS) was estimated at 82%. Patients with indolent lymphoma had an estimated 5-year PFS of 76% and an OS of 92%., Conclusions: DLBCL of the breast was successfully treated with doxorubicin-based chemotherapy alone or with involved field radiotherapy in an estimated 61% of patients at 5 years. A single CNS recurrence was observed in our series of patients, most of whom presented with limited disease., (Copyright (c) 2007 American Cancer Society.)

Rationale: There is currently no available test for monitoring the effect of treatment of latent tuberculosis infection (LTBI) to indicate cure or predict risk of subsequent progression to disease., Objective: We used the T-SPOT.TB assay, which measures T-cell interferon-gamma responses to the Mycobacterium tuberculosis-specific peptides early secretory antigenic target 6-kD protein (ESAT-6) and culture filtrate protein 10 (CFP-10), to determine the effect of LTBI treatment on these responses., Methods: A total of 226 tuberculosis contacts with positive T-SPOT.TB results underwent repeat testing on LTBI treatment completion. The majority (96%) received 6 months of isoniazid. The pre- and post-treatment T-SPOT.TB results were analyzed according to the combined and separate responses to ESAT-6 and CFP-10 antigens., Results: The T-SPOT.TB reverted to negative in 85 (37.6%) contacts at treatment completion. Treatment had a significant effect on the response to CFP-10 (p < 0.001; reversion rate, 48.6%), but not on the response to ESAT-6 (p = 0.081; reversion rate, 21.6%). The median number of spot-forming cells (SFCs)/2.5 x 10(5) peripheral blood mononuclear cells (PBMCs) pre- and post-treatment was 6 versus 4.5 for ESAT-6 (p = 0.116) and 11 versus 4 for CFP-10 (p < 0.001). There was a significant difference between the change (fall) in the pre- and post-treatment responses to CFP-10 (6 SFCs/2.5 x 10(5) PBMCs) and ESAT-6 (0 SFCs/2.5 x 10(5) PBMCs; p < 0.001). Significantly different age-related T-cell responses to the two antigens were found., Conclusion: LTBI treatment had a differential effect on T-cell responses to ESAT-6 and CFP-10 as measured by the T-SPOT.TB. The quantitative response to CFP-10 may be a useful LTBI treatment-monitoring tool.

Dynamic variations in hybridization patterns (rehybridization) were analyzed at B3LYP/6-31G** and MP2/6-31+G* levels. Computations clearly illustrate the generality of rehybridization in a variety of chemical phenomena, which involve structural reorganization in hydrogen-bonded complexes, nonhyperconjugative stereoelectronic effects in saturated heterocycles, Mills-Nixon effect, and contrasting substituent effects in cycloaromatization reactions., (Copyright (c) 2006 Wiley Periodicals, Inc.)

The first total synthesis of (-)-himgaline and a highly enantioselective synthesis of its congener (-)-GB 13 are described. Decarboxylative aza-Michael reaction of the hexacyclic lactone precursor under acidic conditions, followed by basic workup, yielded (-)-GB 13 in 80% yield. Cyclization of (-)-GB 13 to oxohimgaline under acidic conditions, followed by internally coordinated sodium triacetoxyborohydride reduction, gave (-)-himgaline as the exclusive product.

Whereas the reaction of certain propargyl alcohols with TiCl4 in the presence of tertiary alkylamines gives the corresponding chloroallenes in 37-58% yields, reaction with the tertiary arylamines gives the corresponding arylalkynes in 68-77% yields.

The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.

Intramolecular reductive coupling of diimines in the presence of Zn/Ti(O(i)Pr)2Cl2 gives the corresponding (+/-)-2,3-diarylpiperazines in 73-83% yields with dl/meso ratio >99%:<1%. The (+/-)-2,3-diphenylpiperazine obtained in this way was readily resolved partially using L-(+)-tartaric acid, and the enantiomeric purity was enhanced to >99% ee via preparation of hydrogen-bonded salt aggregates using oxalic acid.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

We present a detailed theoretical study of geometries, electronic structure, and energies of transition states and intermediates completing the full Bergman cycloaromatization pathway of ortho-substituted enediynes with a focus on polar and steric contributions to the kinetics and thermodynamics of hydrogen abstraction. This study provides a rare unambiguous example of remote substitution that affects reactivity of a neutral reactive intermediate through an sigma framework.

The tetradentate ligands 1,8-bis(pyrid-2-yl)-3,6-dithiaoctane (pdto) and 1,8-bis(benzimidazol-2-yl)-3,6-dithiaoctane (bbdo) form the complexes [Ru(pdto)(mu-Cl)](2)(ClO(4))(2) 1 and [Ru(bbdo)(mu-Cl)](2)(ClO(4))(2) 2 respectively. The new di-mu-chloro dimers 1 and 2 undergo facile symmetrical bridge cleavage reactions with the diimine ligands 2,2'-bipyridine (bpy) and dipyridylamine (dpa) to form the six-coordinate complexes [Ru(pdto)(bpy)](ClO(4))(2) 3, [Ru(bbdo)(bpy)](ClO(4))(2) 4, [Ru(pdto)(dpa)](ClO(4))(2) 5 and [Ru(bbdo)(dpa)](ClO(4))(2) 6 and with the triimine ligand 2,2':6,2''-terpyridine (terpy) to form the unusual seven-coordinate complexes [Ru(pdto)(terpy)](ClO(4))(2) 7 and [Ru(bbdo)(terpy)](ClO(4))(2) 8. In 1 the dimeric cation [Ru(pdto)(mu-Cl)](2)(2+) is made up of two approximately octahedrally coordinated Ru(II) centers bridged by two chloride ions, which constitute a common edge between the two Ru(II) octahedra. Each ruthenium is coordinated also to two pyridine nitrogen and two thioether sulfur atoms of the tetradentate ligand. The ligand pdto is folded around Ru(II) as a result of the cis-dichloro coordination, which corresponds to a "cis-alpha" configuration [DeltaDelta/LambdaLambda(rac) diastereoisomer] supporting the possibility of some attractive pi-stacking interactions between the parallel py rings at each ruthenium atom. The ruthenium atom in the complex cations 3a and 4 exhibit a distorted octahedral coordination geometry composed of two nitrogen atoms of the bpy and the two thioether sulfur and two py/bzim nitrogen atoms of the pdto/bbdo ligand, which is actually folded around Ru(II) to give a "cis-alpha" isomer. The molecule of complex 5 contains a six-coordinated ruthenium atom chelated by pdto and dpa ligands in the expected distorted octahedral fashion. The (1)H and (13)C NMR spectral data of the complexes throw light on the nature of metal-ligand bonding and the conformations of the chelate rings, which indicates that the dithioether ligands maintain their tendency to fold themselves even in solution. The bis-mu-chloro dimers 1 and 2 show a spin-allowed but Laporte-forbidden t(2g)(6)((1)A(1g))--> t(2g)(5) e(g)(1)((1)T(1g), (1)T(2g)) d-d transition. They also display an intense Ru(II) dpi--> py/bzim (pi*) metal-to-ligand charge transfer (MLCT) transition. The mononuclear complexes 3-8 exhibit dpi-->pi* MLCT transitions in the range 340-450 nm. The binuclear complexes 1 and 2 exhibit a ligand field ((3)MC) luminescence even at room temperature, whereas the mononuclear complexes 3 and 4 show a ligand based radical anion ((3)MLCT) luminescence. The binuclear complexes 1 and 2 undergo two successive oxidation processes corresponding to successive Ru(II)/Ru(III) couples, affording a stable mixed-valence Ru(II)Ru(III) state (K(c): 1, 3.97 x 10(6); 2, 1.10 x 10(6)). The mononuclear complexes 3-7 exhibit only one while 8 shows two quasi-reversible metal-based oxidative processes. The coordinated 'soft' thioether raises the redox potentials significantly by stabilising the 'soft' Ru(II) oxidation state. One or two ligand-based reduction processes were also observed for the mononuclear complexes.