Effect of environmental stress of low pressure on tyrosine aminotransferase and phenylalanine 4-hydroxylase activities in the rat.

Tyrosine aminotransferase activity in the liver increased about fourfold after 9h, on exposure of rats to stress of low pressure. 2. The phenylalanine hydroxylase activity increased about 60% on exposure for 24h or more. 3. An environmental pressure decrease of about 0.033 MN/m2 is needed to increase the activity of tyrosine aminotransferase. 4. Adrenalectomy completely abolished the increase in activity of tyrosine aminotransferase obtained on exposure to low pressure. 5. Treatment with cycloheximide or actinomycin D prevented the increase in activity of tyrosine aminotransferase. 6. Treatment with cycloheximide at the early part of exposure to stress prevented the increase in activity of phenylalanine hydroxylase obtained after 24h.

Purification and properties of Image -mandelate-4-hydroxylase from Pseudomonas convexa

An inducible Image -mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of Image -mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe2+, and O2 for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with Image -mandelate, NADPH, and ferrous sulfate and Km values for these substrates were found to be 1 × 10−4, 1.9 × 10−4, and 4.7 × 10−5 Image , respectively. The enzyme is very specific for Image -mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.

Purification, properties and induction of a specific benzoate-4-hydroxylase from Aspergillus niger (UBC 814)

An inducible benzoate-4-hydroxylase has been partially purified from crude extracts of the mycelial felts of Aspergillus niger. This enzyme catalyzes the transformation of benzoate to p-hydroxybenzoate with equimolar consumption of NADPH and O2. It requires tetrahydropteridine as a prosthetic group. The optimum activity was found at pH 6.2 with a Km value at 30°C of 1.6 · 10−4 M for NADPH and 1.3 · 10−4 M for benzoate. Fe2+ (iron) is required for the enzyme activity. The enzyme is stabilized by the inclusion of benzoate, EDTA and glutathione in the extracting buffer. The enzyme is specific for benzoate as substrate. Sulfhydryl group(s) are essential for enzyme activity as indicated by p-chloromercuri-benzoate and N-ethylmaleimide inactivation. Benzoate-4-hydroxylase activity is decreased in the mycelial felts of Aspergillus niger grown in the presence of higher concentrations of benzoate. Maximum activity of the enzyme was observed at 36 h after inoculation.

A Case Study of the Conformation of Poly(alpha-aminoisobutyric acid): alpha- or 310-Helix

The relative stabilities of a- and Blo-helical structures for polymers of a-aminoisobutyric acid (Aib) have been worked out, using the classical potential energy functions. To make a comparative study, we have used Buckingham "6-exp" and Kitaigorodsky's potential functions. Conformational analysis of the dipeptide segment with Aib residue indicates the necessity for nonplanar distortion of the peptide unit, which is a common feature in the observed crystal structures with Aib residues. In the range of Aw -10 to +loo studied, a-helical conformations are preferred in the region -3" < Aw < +loo, and Blo-helical conformations are preferred in the region -3" > Aw > -10'. Minimum energy conformations for right-handed structures are found in the +ue region of Aw and correspondingly for left-handed structures in the -ue region of Aw. For Aw - 6", a-helical structures have four- or near fourfold symmetry with h - 1.5 A. Such a helix with n = 4 and h = 1.5 A is termed an a'-helix. This structure is found to be consistent with the electron diffraction data of Malcolm3 and energetically more favorable than the standard 310-helix.

LFA-1 Integrin beta2 Chain Phosphorylation Regulates Protein Interactions and Mediates Signals in T Cells

Integrins are heterodimeric transmembrane adhesion receptors composed of alpha- and beta-subunits and they are vital for the function of multicellular organisms. Integrin-mediated adhesion is a complex process involving both affinity regulation and coupling to the actin cytoskeleton. Integrins also function as bidirectional signaling devices, regulating cell adhesion and migration after inside-out signaling, but also signal into the cell to regulate growth, differentiation and apoptosis after ligand binding. The LFA-1 integrin is exclusively expressed in leukocytes and is of fundamental importance for the function of the immune system. The LFA-1 integrins have short intracellular tails, which are devoid of catalytic activity. These cytoplasmic domains are important for integrin regulation and both the alpha and beta chain become phosphorylated. The alpha chain is constitutively phosphorylated, but the beta chain becomes phosphorylated on serine and functionally important threonine residues only after cell activation. The cytoplasmic tails of LFA-1 bind to many cytoskeletal and signaling proteins regulating numerous cell functions. However, the molecular mechanisms behind these interactions have been poorly understood. Phosphorylation of the cytoplasmic tails of the LFA-1 integrin could provide a mechanism to regulate integrin-mediated cytoskeletal interactions and take part in T cell signaling. In this study, the effects of phosphorylation of LFA-1 integrin cytoplasmic tails on different cellular functions were examined. Site-specific phosphorylation of both the alpha- and beta-chains of the LFA-1 was shown to have a role in the regulation of the LFA-1 integrin.Alpha-chain Ser1140 is needed for integrin conformational changes after chemokine- or integrin ligand-induced activation or after activation induced by active Rap1, whereas beta-chain binds to 14-3-3 proteins through the phosphorylated Thr758 and mediates cytoskeletal reorganization. Thr758 phosphorylation also acts as a molecular switch to inhibit filamin binding and allows 14-3-3 protein binding to integrin cytoplasmic domain, and it was also shown to lead to T cell adhesion, Rac-1/Cdc42 activation and expression of the T cell activation marker CD69, indicating a signaling function for Thr758 phosphorylation in T cells. Thus, phosphorylation of the cytoplasmic tails of LFA-1 plays an important role in different functions of the LFA-1 integrin in T cells. It is of vital importance to study the mechanisms and components of integrin regulation since leukocyte adhesion is involved in many functions of the immune system and defects in the regulation of LFA-1 contributes to auto-immune diseases and fundamental defects in the immune system.

Mapping of assembled epitopic regions of human chorionic gonadotropin reveals proximity of CTP alpha to the determinant loop beta 93-100

Three overlapping assembled epitopes of beta hCG have been mapped using MAb probes and a single step solid phase radioimmunoassay. These epitopes have been shown to be at receptor binding region comprising of the loop region beta Cys93-Cys100. Importance of disulphide bonds in maintaining integrity of these epitopes is assessed. Two MAbs (INN 58 and INN 22) interact with the beta region as well as the alpha C-terminal peptide, while the other MAb INN 24 interacts with only the beta region. Cross-reactivity pattern with beta hCG and hLH as web as the reported crystal structure of hCG substantiates the epitope identification. The results demonstrate utility of MAbs as probes in investigations on three-dimensional structure of gonadatropins.

Characterisation of the DNA-polymerase-alpha-primase complex from the silk glands of Bombyx mori

Silk gland cells ofBombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle silk gland nuclei increased by 300000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase with no detectable level of polymerase . DNA polymerase from silk gland extracts was purified to homogeneity (using a series of columns involving ionexchange, gel-filtration and affintiy chromatography), resulting in a 4000-fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180-kDa subunit. The enzyme shows a PI of 6.2 and theKm values for the dNTP vary over 5-16 . The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single-stranded DNA template. The primase activity is resident in the 45-kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase α in silk glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.

Crystal structures and thermal analyses of alkyl 2-deoxy-alpha-D-arabino-hexopyranosides

The crystal structures of alkyl 2-deoxy-alpha-D-arabino-hexopyranosides, with the alkyl chain lengths from C-8 to C-18, are established by the single crystal X-ray structural determination. The even-alkyl chain length derivatives crystallized orthorhombic, with space group P2(1)2(1)2(1), whereas the odd-alkyl chain length derivatives crystallized monoclinic, with space group P2(1). The sugar moieties retained a C-4(1) chair conformation and the conformation of the alkyl chains was all-trans. The molecules formed a bilayer structure, in which alkyl chains were interdigitated.The hydrogen bonds, originating from the sugar moieties, were observed in adjacent layers and also within the same layer, resulting in the formation of infinite chains. The alkyl chains arranged parallel to each other and formed planar structures. The thermal properties of the alkyl 2-deoxy glucosides were analyzed further. It was observed that none of the derivatives exhibited mesomorphism. This study establishes that the absence of the hydroxyl group at C-2 of the sugar moiety results in a non-mesogenic nature of the alkyl 2-deoxy-alpha-D-glycosides, as opposed to the profound mesogenic nature of the normal alkyl glycosides.

A Remarkably Simple One-Step Procedure for the Preparation of alpha-Bromo-alpha,beta-Unsaturated Carbonyl Compounds

An easy and convenient one-step procedure for the conversion of alpha,beta-unsaturated carbonyl compounds into their corresponding bromo-enones using NBS-Et3N center dot 3HBr in the presence of potassium carbonate in dichloromethane at 0 degrees C to room temperature under very mild conditions in high yields and significantly shorter times, is reported.

Ruthenium(II)-CO complexes of N-[(2-pyridyl)methyliden]-alpha(or beta)-aminonaphthalene: Synthesis, spectral studies, crystal structure, redox properties and DFT calculation

The characterization and properties of trans-(X)-[RuX2(CO)(2)(alpha/beta-NaiPy)] (1, 2) (alpha-NaiPy (a), beta-NaiPy (b); X = Cl (1), I (2)) are described in this work. The structures are confirmed by single crystal X-ray diffraction studies. Reaction of these compounds with Me3NO in MeCN has isolated monocarbonyl trans-(X)-RuX2(CO)(MeCN)(alpha/beta-NaiPy)] (3, 4). The complexes show intense emission properties. Quantum yields of 1 and 2 (phi= 0.02-0.08) are higher than 3 and 4 (phi = 0.006-0.015). Voltammogram shows higher Ru(III)/Ru(II) (1.3-1.5 V) potential of 1 and 2 than that of 3 and 4 (0.8-0.9 V) that may be due to coordination of two pi-acidic CO groups in former. The electronic spectra and redox properties of the complexes are compared with the results obtained by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) using polarizable continuum model (CPCM).

The fully implicit stochastic-alpha method for stiff stochastic differential equations

A fully implicit integration method for stochastic differential equations with significant multiplicative noise and stiffness in both the drift and diffusion coefficients has been constructed, analyzed and illustrated with numerical examples in this work. The method has strong order 1.0 consistency and has user-selectable parameters that allow the user to expand the stability region of the method to cover almost the entire drift-diffusion stability plane. The large stability region enables the method to take computationally efficient time steps. A system of chemical Langevin equations simulated with the method illustrates its computational efficiency.

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to +40 mV, with. a dominating fast component and a small slower component. 3. The steady-state inactivation, h(infinity), was related to potential by a Boltzmann distribution, underlying thr ee states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by al? initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O reversible arrow I-1 reversible arrow I-2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m(x) model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha(m) and beta(m), were determined. Their functional dependence on the membrane potential was investigated.

Identification of alpha- and beta-Hydroxy Acid Containing Cyclodepsipeptides in Natural Peptide Mixtures Using Negative Ion Mass Spectrometry

Natural peptide libraries often contain cyclodepsipeptides containing alpha or beta hydroxy residues. Extracts of fungal hyphae of Isaria yield a microheterogenous cyclodepsipeptide mixture in which two classes of molecules can be identified by mass spectral fragmentation of negative ions. In the case of isaridins, which contain an alpha-hydroxy residue and a beta-amino acid residue, a characteristic product ion corresponding to a neutral loss of 72 Da is obtained. hi addition, neutral loss of water followed by a 72 Da loss is also observed. Two distinct modes of fragmentation rationalize the observed product ion distribution. The neutral loss of 72 Da has also been obtained for a roseotoxin component, which is also an alpha-hydroxy residue containing cyclodepsipeptide. In the case of isariins, which contain a beta-hydroxy acid residue, ring opening and subsequent loss of the terminal residue as an unsaturated ketene fragment, rationalizes the observed product ion formation. Fragmentation of negative ions provide characteristic neutral losses, which are diagnostic of the presence of alpha-hydroxy or beta-hydroxy residues.

The alpha method for solving differential algebraic inequality (DAI) systems

This paper describes an algorithm for ``direct numerical integration'' of the initial value Differential-Algebraic Inequalities (DAI) in a time stepping fashion using a sequential quadratic programming (SQP) method solver for detecting and satisfying active path constraints at each time step. The activation of a path constraint generally increases the condition number of the active discretized differential algebraic equation's (DAE) Jacobian and this difficulty is addressed by a regularization property of the alpha method. The algorithm is locally stable when index 1 and index 2 active path constraints and bounds are active. Subject to available regularization it is seen to be stable for active index 3 active path constraints in the numerical examples. For the high index active path constraints, the algorithm uses a user-selectable parameter to perturb the smaller singular values of the Jacobian with a view to reducing the condition number so that the simulation can proceed. The algorithm can be used as a relatively cheaper estimation tool for trajectory and control planning and in the context of model predictive control solutions. It can also be used to generate initial guess values of optimization variables used as input to inequality path constrained dynamic optimization problems. The method is illustrated with examples from space vehicle trajectory and robot path planning.