A Novel Membrane Protein Complex on the Endoplasmic Reticulum and Early Golgi Compartments in the Yeast Saccharomyces cerevisiae

A novel membrane protein, Yml067c in the systematic ORF name, was discovered as a component of immunoisolated vesicles of the early Golgi compartment of the yeast Saccharomyces cerevisiae (Cho et al., FEBS Lett. 469, 151-154 (2000)). Conserved sequences having sequence similarity to Yml067c were widely distributed in the eukaryotes and one of them, Yal042w, was found in the Saccharomyces genome database. In the yeast cell, Yml067c and Yal042w were found to form a heterooligomeric complex by immunoprecipitation of their tagged derivatives from the detergent-solubilized membrane. Cell fractionation and indirect immunofluorescent staining indicated that the majority of these proteins were localized on the ER membrane. Therfore, the Yml067c-Yal042w complex should shuttle between the ER and the early Golgi compartment as well as the p24-family proteins.     

Inhibition of the Bacterial Cell Wall Synthesis in vitro by Enduracidin,a New Polypeptide Antibiotic

Adenosine 5′-phosphosulfate (APS) kinase from a thermophilic bacterium, Bacillus st ear other mophilus, was purified to apparent homogeneity. The apparent molecular weight was 50 kDa, consisting of two 26-kDa subunits. The enzyme was very thermostable and lacked cysteine and methionine residues. Enzyme activity was more stimulated with Mn^{2 +} , Zn^{2 +}, or Co^{2 +} than with Mg^{2 +} and the K_m for ATP and APS were 220 µM and 42 µM, respectively.     

Two oligosaccharides from Marsdenia roylei

Phytochemical analysis of dried twigs of Marsdenia roylei (family Asclepiadaceae) has resulted in the isolation of a trisaccharide, maryal, and a diglycoside, rolinose. Their structures were determined as O-beta-D-oleandropyranosyl-(1-->4)-O-beta-D-digitoxopyranosyl++ +-(1-->4)-D- cymaral and ethyl O-beta-D-oleandropyranosyl-(1-->4)-O-3-O-methyl-6-deoxy-beta-D- allopyranoside, respectively, by chemical degradation and spectroscopic methods.     

The treatment of purified maize oil bodies with organic solvents and exogenous diacylglycerol allows the detection and solubilization of diacylglycerol acyltransferase

In spite of its importance in the biosynthesis of reserve oils in plants, diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) has not been purified to homogeneity, and its study has remained incomplete. We found that the microsomal preparations from developing maize embryos contained substantial amounts of endogenous diacylglycerol (DAG). A solubilization procedure for extracting DAGAT from the microsomes (D. Little, R. Weselake, K. Pomeroy, S.T. Furukawa, J. Bagu, Biochem. J. 304 (1994)) was ineffective in eliminating the endogenous DAG, even after gel filtration. DAG removal through the preparation of acetone powders from the embryos led to the loss of DAGAT activity. Labelled triacylglycerol (TAG) was produced in the standard DAGAT assay when labelled DAG was supplied in benzene solution to the freeze-dried microsomes and the sample was dried and resuspended in an aqueous buffer. In contrast, no labelled TAG was produced when a similar sample supplied with non-labelled DAG was assayed with emulsified labelled DAG and acyl-CoA. Repeated washing of the microsomal freeze-dried fraction with benzene resulted in a complete loss of DAGAT activity in the standard assay, but the activity was restored by the addition of DAG plus phosphatidylcholine or Tween 20 in benzene. Although DAGAT has been reported to be confined mainly to the endoplasmic reticulum, we found that DAGAT activity was high in the purified oil bodies from both developing and mature maize embryos and was not removed by repeated washing with 6 M urea. The DAGAT activity was restored from delipidated oil bodies and from microsomes after the preparations had been resuspended in methanol/acetic acid/water (1:1:1, v/v). Although most of the proteins in the suspension were eluted as a single peak at the void volume after gel filtration chromatography, DAGAT activity was found in later fractions. SDS–PAGE of the peak activity fraction revealed no protein bands after silver staining, and the finding suggest that DAGAT protein is of low abundance and has a high k_cat.     

Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Cytotoxicity of aflatoxin B1 and its chemically synthesised epoxide derivative on the A549 human epithelioid lung cell line

Aflatoxin B₁ (AFB₁) is a carcinogenic mycotoxin found in feeds and in airborne grain dusts. Aflatoxin B₁ requires biotransformation to the AFB₁-8,9 epoxide (AFBO) by a bioactivation system and subsequent covalent binding to DNA or proteins, to exert its carcinogenic potential. The lung contains cytochrome P₄₅₀, prostaglandin-H-synthase, lipoxygenase, epoxide hydrolase and other bioactivation enzymes, and is thus a potential target for the effects of AFB₁ via the routes of inhalation and ingestion. The A549 human epithelioid lung cell line and the methylthiazol tetrazolium (MTT) bioassay were used to investigate the cytotoxicity of AFB₁ and its chemically synthesised epoxide (AFBO) in vitro. Statistical analysis of the MTT results indicated that there were overall significant differences between the control and both the AFB₁-treated (p < 0.0001) and AFBO-treated cells (p = 0.00 2). However, there was no significant difference between AFB₁ and AFBO-treated cells, when the entire range of concentrations were assessed against each other (p = 0.2877). Whenanalysed at each concentration, only at 0.01 mM was there a significant difference between the effects of AFB₁ and AFBO (p = 0.0358). The results of this investigation show that AFB₁ and AFBO are both cytotoxic in the A549 cell line.This revised version was published online in October 2005 with corrections to the Cover Date.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome,Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.