Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes

The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate-induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS-positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space.     

Role of the Nuclease Activities Encoded by Herpes Simplex Virus 1 UL12 in Viral Replication and Neurovirulence

Enzyme-dead mutations in the herpes simplex virus 1 UL12 gene that abolished its endo- and exonuclease activities only slightly reduced viral replication in cell cultures. However, the UL12 null mutation significantly reduced viral replication, suggesting that a UL12 function(s) unrelated to its nuclease activities played a major role in viral replication. In contrast, the enzyme-dead mutations significantly reduced viral neurovirulence in mice, suggesting that UL12 nuclease activities were critical for viral pathogenesis in vivo.     

Frequent overexpression of CADM1/IGSF4 in lung adenocarcinoma

We reported comprehensive screening for antigens (Ags) overexpressed on various carcinomas via isolation of human monoclonal antibodies (mAbs) that may be therapeutic in a previous paper (Proc. Natl. Acad. Sci. USA 105, 7287-7292, 2008). Twenty-one distinct Ags highly expressed on several carcinomas were identified and 356 mAbs with unique sequences turned out to bind to one of the 21 Ags. Among them CADM1/IGSF4 which had been originally referred to as tumor suppressor lung cancer 1 (TSLC1) was included. Therefore we examined the expression of CADM1 in lung cancers in this study. Eight different anti CADM1 mAbs were used for immunohistochemical analysis of 29 fresh lung cancer specimens. Staining patterns were categorized to six groups based on the extent of positive staining and the localization of stained portions. While overexpression of CADM1 was observed on the cell surface of adenocarcinomas at a high frequency, around 60%, positive stainings were rarely observed on that of other lung carcinomas including squamous cell carcinomas. Moreover, some clones among the eight mAbs gave different staining patterns from those by the other clones against the same fresh specimen, suggesting presence of variant forms of CADM1 differentiated by mAbs.     

Oligodendrocytes regulate formation of nodes of Ranvier via the recognition molecule OMgp

The molecular mechanisms underlying the involvement of oligodendrocytes in formation of the nodes of Ranvier (NORs) remain poorly understood. Here we show that oligodendrocyte-myelin glycoprotein (OMgp) aggregates specifically at NORs. Nodal location of OMgp does not occur along demyelinated axons of either Shiverer or proteolipid protein (PLP) transgenic mice. Over-expression of OMgp in OLN-93 cells facilitates process outgrowth. In transgenic mice in which expression of OMgp is down-regulated, myelin thickness declines, and lateral oligodendrocyte loops at the node-paranode junction are less compacted and even join together with the opposite loops, which leads to shortened nodal gaps. Notably, each of these structural abnormalities plus modest down-regulation of expression of Na(+) channel alpha subunit result in reduced conduction velocity in the spinal cords of the mutant mice. Thus, OMgp that is derived from glia has distinct roles in regulating nodal formation and function during CNS myelination.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Synthesis of Collagen-Like Proteins in Embryonic Organs of the Sea Urchin, Hemicentrotus pulcherrimus

In sea urchin embryos exposed to ¹⁴C-proline at 20°C for 3 hr at the gastrula, prism or pluteus stage, ¹⁴C-radioactivity was found in hot acid-extractable proteins, in which more than 4% of the radioactivity was detectable in hydroxyproline residues. In these embryos, ¹⁴C-radioactivity in collagen-like proteins was found in the archenteron, spicule and embryo-wall cells. The rate of synthesis of collagen-like proteins was highest in the archenteron in the mid-gastrula stage, in the embryo-wall cells in the prism stage and in the spicule in the pluteus stage. The rate of synthesis decreased in the archenteron and increased in embryo-wall cells in the period between the mid- and late-gastrula stages, when the rate of synthesis in the spicule was quite low. Thereafter, the rate decreased slightly in the embryo-wall cells, was maintained in archenteron and increased markedly in the spicule. The rates of synthesis of collagen-like proteins are high in these embryonic organs at stages at which development and growth respectively, occur in embryos. Therefore, synthesis of collagen-like proteins probably supports morphogenesis in these embryonic organs.     

Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.