Tum—mutation P35B generates the MHC-binding site of a new antigenic peptide

Immunogenic tumor cell variant P35 was obtained by mutagen treatment of mouse mastocytoma P815. It express a potent new antigen recognized by syngeneic cytolytic T lymphocytes (CTL). This antigen is the result of a point mutation in a gene that is expressed by most healthy cells. A decapeptide encoded by the region spanning the mutation sensitized P815 cells to the relevant CTL, whereas the homologous decapeptide corresponding to the normal sequence did not. Only the mutant decapeptide was capable of enhancing the expression of the D^d-presenting molecule at the cell surface, indicating that the mutation generates a motif which enables the antigenic peptide to bind to D^d. Correspondence to: T. Boon.     

Solubilization and reconstitution of the mitochondrial peptide-sensitive channel

In addition to the voltage-dependent anion channel (VDAC), mitochondrial outer membranes contain a cationic channel of large conductance, which is blocked by a mitochondrial addressing peptide (peptide-sensitive channel, PSC). Bovine adrenal cortex mitochondria were solubilized in 1.5% octyl -glucoside, and membrane vesicles were reconstituted by slow dilution with a low ionic strength buffer. The reconstituted vesicles contained a functional channel possessing the electrical characteristics of the cationic channel, including its sensitivity to the mitochondrial addressing peptide. Important features of the described protocol are the nature of the detergent, its concentration, and the addition of glycerol during the whole procedure. No solubilization could be observed in the presence of cholate.     

The Response to Exercise with Constant Energy Intake in Identical Twins

Seven pairs of young adult male identical twins completed a negative energy balance protocol during which they exercised on cycle ergometers twice a day, 9 out of 10 days, over a period of 93 days while being kept on a constant daily energy and nutrient intake. The total energy deficit caused by exercise above the estimated energy cost of body weight maintenance reached 244 ± 9.8 MJ (Mean ± SEM). Baseline energy intake was estimated over a period of 17 days preceding the negative energy balance protocol. Mean body weight loss was 5.0 kg (SEM = 0.6) (p <0.001) and it was entirely accounted for by the loss of fat mass (p <0.001). Fat-free mass was unchanged. Body energy losses reached 191 MJ (SEM = 24) (p <0.001) which represented about 78% of the estimated energy deficit. Subcutaneous fat loss was slightly more pronounced on the trunk than on the limbs as estimated from skinfolds, circumferences, and computed tomography (CT). The reduction in CT-assessed abdominal visceral fat was quite striking, from 81 cm² (SEM = 5) to 52 cm² (SEM = 6) (p <0.001). At the same submaximal power output level, subjects oxidized more lipids than carbohydrates after the program as indicated by the changes in the respiratory exchange ratio (p <0.05). Intrapair resemblance was observed for the changes in body weight (p <0.05), fat mass (P <0.01), percent fat (p <0.01), body energy content (p <0.01), sum of 10 skinfolds (p <0.01), abdominal visceral fat (p <0.01), fasting plasma triglycerides (p <0.05) and cholesterol (p <0.05), maximal oxygen uptake (p <0.05), and respiratory exchange ratio during submaximal work (p <0.01). We conclude that even though there were large individual differences in response to the negative energy balance and exercise protocol, subjects with the same genotype were more alike in responses than subjects with different genotypes particularly for body fat, body energy, and abdominal visceral fat changes. High lipid oxidizers and low lipid oxidizers during sub-maximal exercise were also seen despite the fact that all subjects had experienced the same exercise and nutritional conditions for about three months.     

IPG (inositolphosphate glycan) as a cellular signal for TGF-β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-β receptors has greatly progressed in the recent years. TGF-β receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-β to its signaling receptors. In addition, four other proteins that bind TGF-β1 or TGF-β2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-β receptors remain an enigma. TGF-β family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-β effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-β1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-β1. Using [³H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-β, supporting that a PI-anchored molecule gives rise to IPG by TGF-β-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-β action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-β on RAC growth. © 1993 Wiley-Liss, Inc.     

Detection and localization of triadin in rat ventricular muscle

Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M_r 102K Ca²⁺ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M_r as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

In vivo morphogenesis and growth characteristics of phages CM (Myoviridae) virulent forRhizobium meliloti

FourRhizobium meliloti bacteriophages belonging to theMyoviridae family of tailed phages were studied. Burst sizes (50–100 virulent particles per infected cell), adsorption rates (2.6–4.1×10^–9 ml/min), and latent periods (2–4 h) made this group heterogeneous. However, these characteristics indicate an important infection ability compared with other rhizobiophages. In vivo morphogenesis of phage CM1, studied by electron microscopy, seems to have steps similar to those of some other tailed phages such as coliphages.     

The ClpE protein involved in biogenesis of the CS31A capsule-like antigen is a member of a periplasmic chaperone family in Gram-negative bacteria

Abstract The putative chaperone-like protein ClpE, required for biogenesis of the Escherichia coli capsule-like antigen CS31A, was compared with ten known periplasmic chaperones from E. coli, Klebsiella pneumoniae, Bordetella pertussis, Haemophilus influenzae and Yersinia pestis . The amino acid sequence alignment was superimposed onto the three-dimensional structure of the PapD chaperone of uropathogenic E. coli , and amino acid residues involved in maintaining the structure integrity of the suggested binding site were found identical in most of the 11 chaperones. Construction of a phylogenetic tree to investigate the relationship within the chaperone family has revealed interesting degrees of relatedness between the different proteins.     

Vigilant Behaviour: Predictability or Randomness? Spectral Analysis of Series of Scan Durations and their Relationship with Inter-scan Intervals

While feeding, many animals frequently look up and visually scan their environment. Using spectral analysis of continuous series of scan durations from a purple sandpiper Calidris maritima and Barbary doves Streptopelia risoria, we show that there are sequential non-random patterns and significant periodicities in all the examined series such that the birds cycled regularly between short and long scans. The cycles are comparable to those for the continuous series of inter-scan intervals of the same behavioural sequences. We suggest a re-examination of the functional costs and benefits of instantaneous randomness versus sequential predictability in alternating between feeding and scanning and a revision of the models of the way animals alternate between these behaviour patterns.     

In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.