Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2K^k and H-2D^d MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2–5)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.     

Effect of elastin peptides on cell proliferation]

Elastin peptides were shown to act on a cell membrane receptor coupled to a G-protein, phospholipase C, and its activation increases IP3 and DAG and opens receptor-dependent Ca(++)-channels. As some growth factors also produce similar modifications in intracellular Ca++, we wanted to explore the effect of elastin peptides on cell proliferation using 3H-thymidine incorporation and cell counting. The concentration of peptides needed for the stimulation of cell proliferation varied between large limits (1 microgram/ml to 10 mg/ml) according to the origin of the cells and the nature of the peptides. The proliferation of CCL 39 chinese hamster lung fibroblasts was enhanced in a dose-dependent fashion in the concentration range of 3 to 10 mg/ml. The proliferation of human skin fibroblasts was enhanced in the concentration range of 0.5 to 3.3 mg/ml and inhibited at higher concentrations. This effect depended little on the average molecular weight (MW) of the peptide preparation, high MW peptides (average 75 kDa) and lower MW peptides (average MW 10 kDa) were both efficient approximately to the same extent. It appears probable that only a small fraction of these peptides possesses this growth promoting property; other sequences might have the opposite effect. The conformation of the peptides may also play an important role. Human sera contain circulating elastin peptides in the concentration range of 1.0 to 10 micrograms/ml, increasing in obstructive arteriopathies and in some hyperlipidemias. It appears therefore that the above findings may have physiopathological significance in the regulation of cell proliferation in normal and pathological conditions.     

Multiple variants in subtelomeric regions of normal karyotypes

We describe a human genomic cosmid clone, 56.1.1, that contains subtelomeric sequences present on multiple human chromosomes. In particular, using fluorescence in situ hybridization, we have identified 16 sites of hybridization on 12 chromosomes. In a sample of 8 unrelated individuals, 10 of these sites showed interindividual variation. Co-hybridization with other polymorphic probes allowed us to demonstrate cytologically heterozygosity at three sites in six individuals. The chromosomal distribution of hybridization sites in a family strongly suggests that these variants are inherited in a Mendelian fashion. These data show that subtelomeric repeats are a rich source of genetic variability. Possible mechanisms of generation of such variants are discussed.     

Overnight (1 mg) dexamethasone suppression testing reliably distinguishes non-cushingoid obesity from Cushing's syndrome.

To determine the sensitivity of the overnight 1-mg dexamethasone suppression test in diagnosing Cushing's syndrome, we evaluated the cortisol responses of 55 subjects (25 non-obese individuals with body mass index less than 25 kg/m2, 20 obese individuals with body mass index greater than 30 kg/m2, and 10 patients with surgically proven Cushing's syndrome) following ingestion of 1 mg dexamethasone at midnight. The basal 8 AM plasma cortisol levels among non-obese and obese individuals and patients with Cushing's syndrome were 310 +/- 85, 377 +/- 91, and 813 +/- 270 nmol/L, respectively. Following 1 mg of dexamethasone, Cushing's syndrome patients showed minimal suppression of cortisol to 609 +/- 180 nmol/L (P = 0.79). Non-obese and obese individuals suppressed to 18.7 +/- 6.0 nmol/L (P less than 0.001) and 22 +/- 7.1 nmol/L (P = 0.003), respectively. The results demonstrated similar cortisol responses to overnight dexamethasone suppression in obese and non-obese groups, and clearly distinguished these subjects from those with Cushing's syndrome. Obesity is not a confounding factor in the 1-mg dexamethasone suppression test.     

Genetic mapping of a gene causing hypertension in the stroke-prone spontaneously hypertensive rat.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a well-characterized model for primary hypertension in humans. High blood pressure in SHRSP shows polygenic inheritance, but none of the loci responsible have previously been identified. To locate genes controlling this quantitative trait, we mapped a large collection of DNA polymorphisms in a cross between SHRSP and the normotensive WKY strain. Here we report strong genetic evidence that a gene, Bp1, having a major effect on blood pressure maps to rat chromosome 10 with a LOD score of 5.10 and is closely linked to the rat gene encoding angiotensin-converting enzyme (ACE), an enzyme that plays a major role in blood pressure homeostasis and is an important target of anti-hypertensive drugs. We also find significant, albeit weaker, linkage to a locus, Bp2, on chromosome 18. We discuss the implications of genetic dissection of quantitative disease-related phenotypes in mammals.     

Potential role of poly(A) polymerase in the assembly of polyadenylation-specific RNP complexes.

To elucidate the mechanism by which poly(A) polymerase functions in the 3'-end processing of pre-mRNAs, polyadenylation-specific RNP complexes were isolated by sedimentation in sucrose density gradients and the fractions were analyzed for the presence of the enzyme. At early stages of the reaction, the RNP complexes were resolved into distinct peaks which sedimented at approximately 18S and 25S. When reactions were carried out under conditions which support cleavage or polyadenylation, the pre-mRNA was specifically assembled into the larger 25S RNP complexes. Polyclonal antibodies raised against the enzyme purified from a rat hepatoma, which have been shown to inhibit cleavage and polyadenylation (Terns, M., and Jacob, S. T., Mol. Cell. Biol. 9:1435-1444, 1989) also prevented assembly of the 25S polyadenylation-specific RNP complexes. Furthermore, formation of these complexes required the presence of a chromatographic fraction containing poly(A) polymerase. UV cross-linking analysis indicated that the purified enzyme could be readily cross-linked to pre-mRNA but in an apparent sequence-independent manner. Reconstitution studies with the fractionated components showed that formation of the 25S RNP complex required the poly(A) polymerase fraction. Although the enzyme has not been directly localized to the specific complexes, the data presented in this report supports the role of poly(A) polymerase as an essential component of polyadenylation-specific complexes which functions both as a structural and enzymatic constituent.     

Uptake and tissue distribution of manganese in the white sucker (Catostomus commersoni) under conditions of low pH

Concentrations of manganese were determined in the liver, kidney, muscle and bone of white suckers (Catostomus commersoni) from five acid (pH < 5.8), and two circumneutral lakes in south-central Ontario. Manganese tissue concentrations were greater in fish captured from the most acidified lakes with the greatest concentrations of dissolved manganese. These fish had increased concentrations of manganese in the liver, as indicated by a comparison of liver:kidney manganese concentration ratios among the seven fish populations. Tissue concentrations of manganese from all populations either were negatively correlated (P < 0.05) or remained constant with fish size indicating homeostatic regulation of this metal. Manganese concentrations of the benthic fauna were positively correlated to sediment concentrations (R=0.30). Lake sediment manganese concentrations were significantly correlated to maximum lake depth (R=0.80, P < 0.03), with the concentrations in the top 0–1 cm dependent on the redox conditions in the seven lakes. Based on the seven lakes studied, manganese concentrations in the benthic-feeding white sucker correlated better with dissolved manganese, than with either the concentrations in food or surficial sediments.     

Cloning, Expression, and Purification of Glutamine Synthetase from Clostridium acetobutylicum

A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg²⁺ in the γ-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.     

Characterization of peptidoglycan stem lengths by solid-state 13C and 15N NMR

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.