A sulfotransferase model: Covalent participation of a macrocyclic oxime in the hydrolysis of 2,4-dinitrophenyl sulfate

The liberation of 2,4-dinitrophenolate ion from 2,4-dinitrophenyl sulfate (DNPS) in aqueous organic solvent with 0.1 N sodium hydroxide was accelerated upon addition of an equimolar amount of Oxime-I (10-hydroxy-11-hydroxyimino[20]-paracyclophane) to the sulfate ester. Oxime-I was found to undergo covalent participation at the oxime group to afford oxime O-sulfonate. The rate acceleration with Oxime-I was larger than that with β-CD (cycloheptaamylose). The catalytic efficiency of Oxime-I has been ascribed primarily to the tighter inclusion of the substrate ester into the more hydrophobic Oxime-I cavity provided by the effective apolar paracyclophane skeleton, as well as to the greater nucleophilicity of the oxime group than of the hydroxyl group in β-CD. Consequently, Oxime-I may be considered as a conventional model for arylsulfatases and sulfotransferases, providing the effective binding process for the substrate.     

Studies of ANF processing and secretion using a primary cardiocyte culture model.

In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion.     

Real-Time Monitoring of Slow-Wave Sleep by Electroencephalogram Variance

Based on statistical variance as an index of electroencephalogram (EEG) parameters, we monitored slow-wave sleep in both humans and rats in real time and on-line with a widely used personal computer. This EEG variance method may be a useful tool to carry out biological rhythm research, including sleep studies.     

Observations on the effect of visual and olfactory ablation on the swimming behavior of migrating adult chum salmon,Oncorhynchus keta

The behavior of chum slamon,Oncorhynchus keta, was studied using ultrasonic telemetry in the waters off the Okhotsk coast of Hokkaido from 1979 to 1981. Thirty-six adult fish were outfitted with a 50 KHz ultrasonic transmitter provided with either a depth sensor, depth/illumination sensors, or depth/ temperature sensors. Twenty-one of the experimental fish were used as controls and left intact. Of the remaining fish, six had their sight destroyed, seven had their olfactory nerves severed or their nares filled with wax, one had both senses destroyed, and one had a sham operation, but had only a shallow transverse cut made in the skin over the olfactory nerves. Control fish and the fish with the sham operation initially swam in a horizontal zigzag pattern while fish with an obliterated sense swam in a similar pattern but to a reduced extent. The horizontal and vertical speeds of the intact fish were faster than those of the fish with a destroyed sense. Amplitude of vertical movements of the intact fish stretched from surface to bottom, while fish with destroyed vision tended towards midwater. Fish without an olfactory sense tended to be at the surface or near the bottom. Regular vertical movement would be effective in refreshing olfactory epithelia which might become acclimated when exposed to one odor. The vertical movements can be found in the horizontal zigzag movements in coastal and near shore migrations, as well as in the stream phase of homing. The zigzag movements, both horizontal and vertical, allow the fish to sense the water masses and locate the correct tributary.     

Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.     

Implication of Progranulin and C1q/TNF-Related Protein-3 (CTRP3) on Inflammation and Atherosclerosis in Subjects with or without Metabolic Syndrome

Objective

Progranulin and C1q/TNF-related protein-3 (CTRP3) were recently discovered as novel adipokines which may link obesity with altered regulation of glucose metabolism, chronic inflammation and insulin resistance.

Research Design and Methods

We examined circulating progranulin and CTRP3 concentrations in 127 subjects with (n = 44) or without metabolic syndrome (n = 83). Furthermore, we evaluated the relationship of progranulin and CTRP3 levels with inflammatory markers and cardiometabolic risk factors, including high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), estimated glomerular filtration rate (eGFR), and adiponectin serum concentrations, as well as carotid intima-media thickness (CIMT).

Results

Circulating progranulin levels are significantly related with inflammatory markers, hsCRP (r = 0.30, P = 0.001) and IL-6 (r = 0.30, P = 0.001), whereas CTRP3 concentrations exhibit a significant association with cardiometabolic risk factors, including waist circumference (r = −0.21), diastolic blood pressure (r = −0.21), fasting glucose (r = −0.20), triglyceride (r = −0.34), total cholesterol (r = −0.25), eGFR (r = 0.39) and adiponectin (r = 0.26) levels. Serum progranulin concentrations were higher in patients with metabolic syndrome than those of the control group (199.55 [179.33, 215.53] vs. 185.10 [160.30, 204.90], P = 0.051) and the number of metabolic syndrome components had a significant positive correlation with progranulin levels (r = 0.227, P = 0.010). In multiple regression analysis, IL-6 and triglyceride levels were significant predictors of serum progranulin levels (R ² = 0.251). Furthermore, serum progranulin level was an independent predictor for increased CIMT in subjects without metabolic syndrome after adjusting for other cardiovascular risk factors (R ² = 0.365).

Conclusions

Serum progranulin levels are significantly associated with systemic inflammatory markers and were an independent predictor for atherosclerosis in subjects without metabolic syndrome.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01668888","term_id":"NCT01668888"}}NCT01668888     

Determination of the Role of DDX3 a Factor Involved in Mammalian RNAi Pathway Using an shRNA-Expression Library

RNA interference (RNAi) is an endogenous RNA-destruction phenomenon induced by certain double-stranded RNAs (dsRNAs). In RNAi, dsRNAs are processed into small interfering RNAs (siRNAs) which in turn trigger the cleavage of the target mRNA. Here, using a short hairpin RNA-expression library, we identified a DEAD-box helicase 3, DDX3, as an essential factor involved in RNAi pathway and revealed that DDX3 is colocalized with Ago2, an essential factor in RNAi pathway that cleaves target mRNA. Results of experiments with a dominant negative mutant of DDX3 further confirmed that this factor affects the RNAi activity. Together, DDX3 functions to assure mammalian RNAi pathway. Together, our results indicate that DDX3 is a new key molecule to understand the molecular mechanism underlying RNAi pathway in mammals.     

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.     

The Spherical Expansion of H2 Dimer Interaction Energy by Double Symmetry-Adapted Perturbation Theory

Abstract

The weak interaction energy of H₂ dimer is studied by double symmetry-adapted perturbation theory (SAPT) within second-order of intermolecular and intramonomer perturbation for molecular simulations. The assumed orientations of H₂ dimer are linear, parallel, T type and X type. Among four orientations T orientation is the most stable, while linear orientation is the most repulsive. The second-order dispersion energy E _disp ⁽²⁾ is the most attractive contribution in all orientations. The interaction energy has the anisotropy, so we expressed our total interaction energy by the spherical expansion to compare with the experimental value. The isotropic interaction energy is about 85% of the experimental value.     

Intra‐specific pollen polymorphism in Mimosa pigra (Mimosaceae)

Five different types of pollen tetrads were detected from the short and long stamens of the male and hermaphrodite flowers of the androdioecious species, Mimosa pigra L. Light (LM) and scanning electron microscopes (SEM) were used in this study to redescribe the pollen forms and to identify the various pollen types encountered. An identification key and relative percentages of these pollen types were also presented. The study showed that the long stamens of the male flowers and those of the hermaphrodite ones have five (I, II, III, IV and V) and two (I and V) pollen types respectively, whereas the short stamens of both male and hermaphrodite flowers have only one pollen type (type I). We report here the first incidence of intra‐specific pollen polymorphism in androdioecious species.