Mechanisms of transmembrane signalling in human T cell activation

Several monoclonal antibodies directed against a number of T cell surface molecules are used to elucidate the role of these molecules (cell surface molecules) in T cell activation. The activation of T cells via these molecules are both antigen-dependent (CD3/TcR complex) and antigen-independent. Irrespective of their antigen-dependency, these monoclonal antibodies activate T cells by a classical signal transduction pathway, in which the binding of monoclonal antibodies to their cell surface receptors leads to activation of phospholipase C resulting in the the depolarization of plasma membrane, hydrolysis of IP2 and IP3 and DAG, the second messengers. IP3 leads to mobilization of intracellular calcium to contribute to an increase in [Ca⁺⁺]_i, whereas DAG causes activation and translocation of PKC and an increasing apparent affinity for Ca⁺⁺. The role of IN in the mobilization of intracellular calcium is emerging. In addition, influx of extracellular calcium also contributes to increase in [Ca^–+];. The increase in [Ca⁺⁺]; following activation via some T cell surface antigen is predominantly due to intracellular mobilization of Ca^–+ (e.g. CD3/TcR complex), whereas activation via other T cell surface antigen, the increase in [Ca^+–]_i is almost entirely due to an influx of extracellular calcium (e.g. CD5 antigen). All these molecules activate autocrine system of T cell growth, namely IL-2 production, IL-2 receptor expression and T cell proliferation.     

Effect of DDT on hepatic mixed-function oxidase activity in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks resulted in significant decrease in the body weight and marked reduction in the hepatic vitamin A content. The levels of hepatic phase I microsomal enzymes cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase were found to be substantially reduced by vitamin A-deficiency. Also, the activity of phase II microsomal UDP - glucuronyl transferase enzyme was significantly decreased in deficient animals. Following repeated oral administration of DDT (15 mg/kg/body wt/day) for 21 days, the phase I microsomal enzymes were induced to a greater extent in controls as compared to deficient animals. UDP - glucuronyltransferase remained insensitive to DDT induction. The results imply that the capacity for induction of the hepatic mixed-function oxidase enzyme system is impaired in deficient animals concurrently exposed to DDT.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.     

A novel primase-free form of murine DNA polymerase alpha induced by infection with minute virus of mice

Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.     

Role of endogenous opioids and histamine in morphine induced emesis

The role of opioid and histaminergic system in morphine induced emesis was investigated in dogs. Morphine (25 micrograms, icv) consistently evoked emesis with an average latency of 195 +/- 29 sec which was fully accounted for by an action on the chemoreceptor trigger zone (CTZ) as its ablation rendered animals refractory to vomiting. Intraventricular pretreatment with opioid antagonist naloxone, histamine H1 antagonist mepyramine and H2 antagonists metiamide and cimetidine afforded protection to icv morphine emesis. The CSF histamine concentration was significantly raised 5 min after icv morphine administration. The results suggest that both endogenous opioid and histamine are involved in morphine emesis. Naloxone in high doses (1600 micrograms, icv) elicited emesis which was not blocked by CTZ ablation confirming our earlier report.     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Energetics of the hairpin to mismatched duplex transition of d(GCCGCAGC) on NaCl solution

We report Potential of Mean Force studies to describe the relative thermodynamic stabilities of d(GCCGCAGC) in a mismatched duplex and a hairpin monomer conformation in NaCl solution. The PMF calculations are combined with previous molecular mechanics and normal mode analysis in order to estimate the role of different components of the free energy in determining the relative stability of the duplex and hairpin structures. The high entropy associated with the loop region and the lack of minor groove phosphate-phosphate interactions in the hairpin compete against the gain in enthalpic contribution to the free energy due to base pairing in the mismatched duplex. The combined free energy calculations show that the hairpin is the most stable conformation at low salt and that a hairpin to duplex transition takes place at approximately 0.47 M NaCl. In addition, we studied the hairpin to partially stacked single helical conformation equilibrium at low salt. We found a small variation in transition temperature in salt concentration, delta Tm/delta log10(cs) approximately 2-3 degrees K/decade, in contrast to the duplex to hairpin or duplex to partially stacked single helix transition where the transition temperature exhibited marked dependence on salt concentration. This is in qualitative agreement with experimental data. Based on the Potential of Mean Force free energy calculation, the order of relative stability of the three-conformations studied varies with salt concentration. We observed the following orders of stability: stacked single helix greater than hairpin greater than duplex for cs less than 0.77 M NaCl; single helix greater than duplex greater than hairpin for 0.77 less than Cs less than 2.1 M; and duplex greater than hairpin greater than single strand for cs greater than 2.1 M. From the calculated PMF free energy curves in the NaCl concentration range, 0.012 less than cs less than 5.0 M, we can assign upper and lower bounds for the non-ionic differences in free energy between the duplex, hairpin, and stacked single helical states (at standard conditions: cs = 1.0 M, T = 25 degrees C, and 1 M oligomer concentration). We found that for delta G duplex single helix = G duplex - 2 x G single helix less than -7.38 Kcal/mol, the single helix is the least stable state. For the duplex-to-hairpin free energy difference in the range, -1.87 less than delta G duplex-hairpin less than 0.03 Kcal/mol, there will always be a salt-induced hairpin-to-duplex transition for 0.01 less than cs less than 1.6 M NaCl. If delta G duplex-hairpin less than -1.87, the duplex is always more stable than the hairpin; and for delta G duplex-hairpin greater than Kcal/mol, the hairpin state is always more stable than the duplex, for all salt concentrations.     

Degradation of synthetic lignins and some lignin monomers by the yeastRhodotorula glutinis

Rhodotorula glutinis degraded variously¹⁴C-labelled synthetic lignins in the presence of 0.1% glucose as co-substrate. Side chain-labelled DHP was degraded the most. While this yeastutilized vanillate and forulate for growth, sinapate/syringate were poorly or not degraded. Gallate, protocatechuate and acetate also supported growth. [carboxy-¹⁴C]Syringate was rapidly converted to¹⁴CO₂ by the yeast only in presence of glucose while [carboxy-¹⁴C]vanillate did not require any additional cosubstrate for mineralization. Ring-labelled vanillyl alcohol was also dagraded proving that the yeast could rapidly metabolize guaiacyl structures while syringyl structures required the presence of additional energy sources.

---

Résumé Rhodotorula glutinis dégrade des lignines synthétiques marquées au¹⁴C de manière variée, en présence de 0.1% de glucose comme co-substrat. La DHP marquée sur la chaîne latérale est dégradée le plus. Alors que cette levure utilise le vanillate et le férulate pour sa croissance, le sinapate et le syringate sont peu ou prou dégradables. Le gallate, le protocatéchuate et l'acétate supportent également la croissance. Le syringate marqué au¹⁴C dans sa fonctioncarboxyle estrapidement convert en¹⁴CO₂ par le levure mais exclusivement en présence de glucose, tandis que le vanillate marqué au¹⁴C dans sa fonction carboxyle ne requiert aucun co-substrat additionel pour sa minéralisation. L'alcool vanillique marqué dans son cycle, est également dégradé démonstrant ainsi que la levure peut métaboliser rapidement des structures guaiacyliques tandis que les structures syringiques requièrent la présence de sources auxiliaires d'énergie.