Construction of a plasmid for high-level expression of goat alpha-lactalbumin and its derivative in E. coli

The high-level expression system of goat alpha-lactalbumin (alpha-LA) in E. coli was established by fusing the alpha-LA cDNA to porcine adenylate kinase cDNA and expressing the fused gene under the control of tac promoter. For high-level expression, elimination of 3'-noncoding region of the alpha-LA cDNA was found to be necessary.     

Interleukin 1-like factors can accumulate 5-hydroxytryptamine in the liver of mice and can induce hypoglycaemia

After the injection into mice of culture medium of P388D1 cells, a murine macrophage cell line, 5-hydroxytryptamine accumulated in the liver and blood glucose declined. The factors capable of inducing these responses were purified by gel filtration and chromatofocusing. With these procedures, the activity to induce the increase in 5-hydroxytryptamine in the liver accompanied the activity to induce hypoglycaemia. Moreover, through the purification, the factors were found in the fraction of interleukin 1, a lymphocyte-activating factor. These results suggest that the factors capable of inducing the increase in 5-hydroxytryptamine and hypoglycaemia are likely to be interleukin 1 molecules or molecules closely related to interleukin 1. The present and previous findings together with those in the literature support the idea that the increase in 5-hydroxytryptamine in the liver might be a cause of hypoglycaemia. These findings may provide new and important information about the roles of macrophages in inflammation or in immune responses.     

Properties of aspartate racemase, a pyridoxal 5'-phosphate-independent amino acid racemase.

Aspartate racemase from Streptococcus thermophilus contains no pyridoxal 5'-phosphate or other cofactors such as FAD, NAD+, and metal ions. It was affected by neither carbonyl reagents such as hydroxylamine nor sodium borohydride but was strongly inhibited by iodoacetamide and other thiol reagents. Aspartate, cysteate, and cysteine sulfinate were the only substrates. The Km values for L- and D-aspartate were 35 and 8.7 mM, respectively. The enzyme catalyzed the exchange of alpha-hydrogen of the substrate with the solvent hydrogen. Racemization of L-aspartate in 2H2O showed an overshooting in the optical rotation of aspartate before the substrate was fully racemized. This shows that the removal of alpha-hydrogen of the substrate is at least partially rate-determining. When L- or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer. The results strongly suggest that aspartate racemase contains two hydrogen acceptors.     

An Interatomic Potential Model for H2O: Applications to Water and Ice Polymorphs

Abstract

A new interatomic potential model for H₂O which consists of 2-body central (O-H, O-O and H-H) and 3-body teams and does not contain artificial constraints on the motions of oxygen and hydrogen atoms is proposed. The interatomic potential function parameters were determined empirically so as to reproduce the fundamental and essential features of water and ice Ih using molecular dynamics (MD) methods.

We carried out the MD simulations of water, and find the Ice Ih, Ice II and Ice IX using this potential model, in structures and physical properties are in agreement with the experimental results except for the compressibilities of both water and ice Ih. We expect that, by refining this model, we can apply this model to problems involving the reaction of water molecules with other components.     

The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.

The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.     

Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.