Regulation of perforin activation and pre‐synaptic toxicity through C‐terminal glycosylation

Perforin is a highly cytotoxic pore‐forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca²⁺‐rich endoplasmic reticulum, remains unknown. Here, we show that N‐linked glycosylation of the perforin C‐terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C‐terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C‐terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post‐translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.     

Suppression of ongoing disease in a nonhuman primate model of multiple sclerosis by a human-anti-human IL-12p40 antibody

IL-12p40 is a shared subunit of two cytokines with overlapping activities in the induction of autoreactive Th1 cells and therefore a potential target of therapy in Th1-mediated diseases. We have examined whether ongoing disease in a nonhuman primate model of multiple sclerosis (MS) can be suppressed with a new human IgG1kappa Ab against human IL-12p40. Lesions developing in the brain white matter were visualized and characterized with standard magnetic resonance imaging techniques. To reflect the treatment of MS patients, treatment with the Ab was initiated after active brain white matter lesions were detected in T2-weighted images. In placebo-treated control monkeys we observed the expected progressive increase in the total T2 lesion volume and markedly increased T2 relaxation times, a magnetic resonance imaging marker of inflammation. In contrast, in monkeys treated with anti-IL-12p40 Ab, changes in the total T2 lesion volume and T2 relaxation times were significantly suppressed. Moreover, the time interval to serious neurological deficit was delayed from 31 +/- 10 to 64 +/- 20 days (odds ratio, 0.312). These results, in a disease model with high similarity to MS, are important for ongoing and planned trials of therapies that target IL-12 and/or IL-23.     

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity

Understanding the selectivity of aquaporin water channels will require structural and functional studies of wild-type and modified proteins; however, expression systems have not previously yielded aquaporins in the necessary milligram quantities. Here we report expression of a histidine-tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous expression system. 10-His-AqpZ is solubilized and purified to near homogeneity in a single step with a final yield of approximately 2.5 mg/l of culture. The histidine tag is removed by trypsin, yielding the native protein with the addition of three N-terminal residues, as confirmed by microsequencing. Sucrose gradient sedimentation analysis showed that the native, solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other known aquaporins, AqpZ tetramers are not readily dissociated by 1% SDS at neutral pH. Hydrophilic reducing agents have a limited effect on the stability of the tetramer in 1% SDS, whereas incubations for more than 24 hours, pH values below 5.6, or exposure to the hydrophobic reducing agent ethanedithiol cause dissociation into monomers. Cys20, but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS. Upon reconstitution into proteoliposomes, AqpZ displays very high osmotic water permeability (pf > or = 10 x 10(-14) cm3 s-1 subunit-1) and low Arrhenius activation energy (Ea = 3.7 kcal/mol), similar to mammalian aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was detected. Expression of native and modified AqpZ in milligram quantities has permitted biophysical characterization of this remarkably stable aquaporin tetramer, which is being utilized for high-resolution structural studies.     

Glucosidase II, a protein of the endoplasmic reticulum with high mannose oligosaccharide chains and a rapid turnover

Glucosidase II is regarded as a resident protein of the endoplasmatic reticulum. The enzyme removes alpha-1-3-linked glucose from high mannose oligosaccharides N-linked to asparagine residues of glycoproteins. Monospecific antibodies raised against the pig kidney enzyme are used to study the metabolism of the enzyme in a rat hepatoma cell line. These antiglucosidase II antibodies specifically immune precipitate glucosidase II as a 100,000-Da species from [35S]methionine-labeled cells. In addition, protein blotting and immune staining of cell extracts from both rat liver and human and rat hepatoma cell lines show identity in apparent Mr (100,000). Glucosidase II synthesized in the presence of tunicamycin is approximately 94,000 Da, indicating the presence of one or more N-linked oligosaccharide chains. Cell-free protein synthesis of rat hepatoma total RNA demonstrates that glucosidase II is synthesized as a slightly higher molecular weight species as compared to the polypeptide synthesized in whole cells in the presence of tunicamycin, indicating that the enzyme has a cleavable signal sequence. Using a pulse-chase protocol, the apparent molecular weight does not change upon longer chase periods. In addition, the 100,000-Da protein remains sensitive to endo-beta-N-acetylglucosaminidase H regardless of prolonged chase periods. The cells incorporate [3H]mannose into the enzyme; after release with endo-beta-N-acetylglucosaminidase H, most of the radioactivity comigrates with Glc1-Man9-GlcNAc on a gel filtration column. Phase separation in Triton X-114 shows a partition between the aqueous and the Triton phase, the major portion being separated in the aqueous phase. In rat hepatoma cells glucosidase II has a half-life of 50 min. This value is not altered if the cells are grown in the presence of monensin nor of methyl-deoxynoijirimycin. However, tunicamycin and low concentrations or primaquine (raising the pH of acidic compartments) causes a 100% increase in half-life of glucosidase II. We conclude that glucosidase II is a hydrophilic, probably not a transmembrane membrane, protein with a short half-life. It is the first example of an oligosaccharide-processing enzyme not being an integral membrane protein.     

Optical characterization of Photosystem II electron donors

Detailed absorbance difference spectra are reported for the Photosystem II acceptor Q, the secondary donor Z, and the donor involved in photosynthetic oxygen evolution which we call M. The spectra of Z and Q could be resolved by analysis of flash-induced kinetics of prompt and delayed fluorescence, EPR signal II_f and absorbance changes in Tris-washed system II preparations in the presence of ferricyanide and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The spectrum of Z oxidation consists mainly of positive bands at 260, 300 and 390–450 nm on which a chlorophyll a band shift around 438 nm is superimposed, and is largely pH-independent as is also the case for the spectrum of Q reduction. The re-reduction of Z⁺ occurred in the millisecond time range, and could be explained by a competition between back reaction with Q^? (120 ms at pH 6.0) and reduction by ferrocyanide. When the Tris treatment is omitted the preparations evolve oxygen, and the photoreduction of Q (with DCMU present) is accompanied by the oxidation of M. The Q spectrum being known, the spectrum of the oxidation of M could be determined as well. It consists of a broad, asymmetric increase peaking near 305 nm and of a Chl a band shift, which is about the same as that accompanying Z in Tris-washed system II. Comparison with spectra of model compounds suggests that Z is a bound plastoquinol which is oxidized to the semiquinone cation and that the oxidation of M is an Mn(III) → Mn(IV) transition.     

Stable carbon and oxygen isotopes in human tooth enamel: Identifying breastfeeding and weaning in prehistory. Am. J. Phys. Anthropol. 106: 1–18.

ERRATUM: Wright LE and Schwarcz HP (1998) Stable Carbon and Oxygen Isotopes in Human Tooth Enamel: Identifying Breastfeeding and Weaning in Prehistory. Am. J. Phys. Anthropol. 106: 1–18. Isotopic ratios were incorrectly printed as percentages (%) rather than units permil (‰). Wherever “breast feeding” and “breast fed” occur, the words should be combined into “breastfeeding” and “breastfed” respectively. The correct information on isotopic ratios is the following: p. 1, Abstract: all % signs should be “‰”; p. 2, column two, last line: “… δ¹³C values¹, have been …”; p. 2, Footnote 1 should read: “Isotope ratios of carbon and oxygen are expressed in δ notation as follows, δ = [(R_sample/R_standard) − 1] × 1000, where R = ¹³C/¹²C for δ¹³C, and R = ¹⁸O/¹⁶O for δ¹⁸O, and are in units permil, ‰.”; p. 3, column two: all % signs should be “‰”, except for the 23rd line from the bottom, which reads: “… provided 95% of water intake by all infants …”; p. 5: all % signs should be “‰”; p. 6, column one: 8th and 9th line from bottom should be “… mean deviations were 0.029‰ for δ¹³C and 0.024‰ for δ¹⁸O …”; p. 6, column one: 2nd line from bottom should be “… only 0.208‰ for δ¹³C and only 0.091‰ for δ¹⁸O …”; p. 6, column two: top line: “… of 0.5‰ in δ¹³C and 0.2‰ in δ¹⁸O …”; p. 8, 9 and 10: all % signs should be “‰”; p. 11, column two: 7th line from top: “… the lipids are 4-6‰ lighter …”; p. 12, 13 and 14: all % signs should be “‰.”     

The hydrogenosomes of Psalteriomonas lanterna

Background  

Hydrogenosomes are organelles that produce molecular hydrogen and ATP. The broad phylogenetic distribution of their hosts suggests that the hydrogenosomes of these organisms evolved several times independently from the mitochondria of aerobic progenitors. Morphology and 18S rRNA phylogeny suggest that the microaerophilic amoeboflagellate Psalteriomonas lanterna, which possesses hydrogenosomes and elusive "modified mitochondria", belongs to the Heterolobosea, a taxon that consists predominantly of aerobic, mitochondriate organisms. This taxon is rather unrelated to taxa with hitherto studied hydrogenosomes.     

Genetic lesions within the 3a gene of SARS-CoV

Phage N5 is one of the phages of Vibrio cholerae serovar O1 biotype El Tor (Ghosh, A. N., Ansari, M. Q., and Dutta, G. C. Isolation and morphological characterization of El Tor cholera phages. J. Gen. Virol. 70: 2241–2243, 1989). In the present communication the growth curve, molecular weight and confirmation of the genome, partial denaturation map and restriction endonuclease digestion pattern have been determined. Partial denaturation map indicates that the genome has non-permuted / invariant sequence. Presence of cohesive ends has also been documented.