PRMT 3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation

Methylation is one of the many post-translational modifications that modulate protein function. Although asymmetric NG,NG-dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine N-methyltransferases (PRMT), has been known for some time, members of this enzyme class have only recently been cloned. The first example of this type of enzyme, designated PRMT1, cloned because of its ability to interact with the mammalian TIS21 immediate-early protein, was then shown to have protein arginine methyltransferase activity. We have now isolated rat and human cDNA orthologues that encode proteins with substantial sequence similarity to PRMT1. A recombinant glutathione S-transferase (GST) fusion product of this new rat protein, named PRMT3, asymmetrically dimethylates arginine residues present both in the designed substrate GST-GAR and in substrate proteins present in hypomethylated extracts of a yeast rmt1 mutant that lacks type I arginine methyltransferase activity; PRMT3 is thus a functional type I protein arginine N-methyltransferase. However, rat PRMT1 and PRMT3 glutathione S-transferase fusion proteins have distinct enzyme specificities for substrates present in both hypomethylated rmt1 yeast extract and hypomethylated RAT1 embryo cell extract. TIS21 protein modulates the enzymatic activity of recombinant GST-PRMT1 fusion protein but not the activity of GST-PRMT3. Western blot analysis of gel filtration fractions suggests that PRMT3 is present as a monomer in RAT1 cell extracts. In contrast, PRMT1 is present in an oligomeric complex. Immunofluorescence analysis localized PRMT1 predominantly to the nucleus of RAT1 cells. In contrast, PRMT3 is predominantly cytoplasmic.     

Expression of beta 2-integrins and L-selectin on polymorphonuclear leukocytes in septic patients

Adhesion molecules on polymorphonuclear leukocytes (PMNL) play an important role in nonspecific defense mechanisms directed at invading microorganisms. When local infection, however, cannot be controlled, a systemic inflammatory response syndrome (SIRS) ensues which may progress to septic shock and multiple organ failure, these being major determinants of the patient's outcome. In the present study, the expression of beta 2-integrins and L-selectin on blood PMNL was measured on subsequent days in patients with sepsis (n = 17) and in healthy volunteers (n = 15). beta 2-Integrins and L-selectin molecules were detected by flow cytometry, using the monoclonal antibodies IB4 (anti-CD18) and Dreg200 (anti-CD62L), respectively. Adhesion molecules were determined at baseline immediately after blood collection and also 45 min after incubation of cells in vitro at body temperature to allow for spontaneous regulation. In addition, PMNL were activated by receptor-dependent and receptor-independent stimuli to characterize stimulus-specific adhesion molecule expression. In parallel with the measurement of adhesion molecules, severity of sepsis was assessed by the Elebute score. The results demonstrate significant differences in the basal, spontaneous and stimulus-induced expression of adhesion molecules between healthy volunteers, survivors (n = 11) and nonsurvivors (n = 6). Moreover, when survivors and nonsurvivors with severe sepsis (Elebute score > 12) were compared, basal expressions of both beta 2-integrins and L-selectin were significantly lower in patients who did not survive. Thus, measurement of adhesion molecules on circulating PMNL may be useful to identify septic patients at high risk for lethal outcome.     

All-trans beta-carotene is absorbed preferentially to 9-cis beta-carotene, but the latter accumulates in the tissues of domestic ferrets (Mustela putorius puro)

The algae Dunaliella bardawil and Dunaliella salina naturally contain large concentrations of all-trans and 9-cis beta-carotene (betaC). The purpose of this study was to compare the relative serum and tissue accumulation of all-trans and 9-cis betaC in ferrets fed different ratios of all-trans/9-cis betaC derived from two commercial sources, D. bardawil or D. salina (Betatene). Male ferrets (7 wk old) were fed carotene-free, pelleted diets for 27 d. Beginning on d 18, groups of ferrets (n = 6 or 7) received daily, one of six oral supplements varying in ratios of 9-cis and all-trans betaC mixed with approximately 1.0mL of Ensure. Four supplements containing 5.2-8.3 micromol total betaC were prepared from a 20% Betatene preparation, D. bardawil, a high-cis Betatene preparation, and Betatene further enriched in 9-cis betaC with all-trans betaC/9-cis betaC ratios of 2.2, 1.5, 0.6 and 0.4, respectively. Two control supplements, high and low betaC, were prepared from commercial betaC beadlets. The high control supplement had an all-trans/9-cis ratio of 19.0, whereas 9-cis betaC was not detected in the low supplement. On d 27, serum and tissues were obtained for HPLC analysis of betaC and its isomers. Analysis of livers showed that all-trans betaC was the primary isomer present, but 9-cis and other isomers were also detected in all groups. The hepatic all-trans/9-cis ratios were 5.9, 4.9, 2.5, 1.4, 52.2 and47.5, respectively, for the groups listed above. Lower amounts of all-trans and 9-cis betaC were found in kidneys compared with the liver, but ratios of all-trans/9-cis were not different among groups. Only trace amounts of 9-cis betaC were found in serum. These results demonstrate that the algae D. bardawil and D. salina provide a bioavailable source of betaC isomers, but, as in humans, absorption of 9-cis betaC is poor and any 9-cis betaC absorbed is apparently cleared by the liver.     

Holistic Information Security: ISO 27001 and Due Care

“The only truly secure system is one that is powered off, cast in a block of concrete and sealed in a lead-lined room with armed guards—and even then I have my doubts.”

—Gene Spafford, Professor of Computer Science, Purdue University     

Striate cortex extracts higher-order spatial correlations from visual textures

Spatial correlations define the statistical structure of any visual image. Two-point correlations inform the visual system about the spatial frequency content of an image. Higher-order correlations can capture salient features such as object contours. We studied "isodipole" texture discrimination in V1 to determine if higher-order spatial correlations can be extracted by early stages of cortical processing. We made epicortical, local field potential, and single-cell recordings of responses elicited by isodipole texture interchange in anesthetized monkeys. Our studies demonstrate that single neurons in V1 can signal the presence of higher-order spatial correlations in visual textures. This places a computational mechanism, which may be essential for form vision at the earliest stage of cortical processing.     

Incremental value of prognostic testing in patients with known or suspected ischemic heart disease: a basis for optimal utilization of exercise technetium-99m sestamibi myocardial perfusion single-photon emission computed tomography

OBJECTIVES: This study assessed the incremental prognostic implications of normal and equivocal exercise technetium-99m (Tc-99m) sestamibi single-photon emission computed tomography (SPECT) and sought to determine its incremental prognostic value, impact on patient management and cost implications. BACKGROUND: The prognostic implications of Tc-99m sestamibi SPECT are not well defined, and risk stratification using this test has not been explored. METHODS: We studied 1,702 patients referred for exercise Tc-99m sestamibi SPECT who were followed up for a mean (+/- SD) of 20 +/- 5 months. Patients with previous percutaneous transluminal coronary angioplasty or coronary artery bypass surgery were excluded. The SPECT studies were assessed using semiquantitative visual analysis. Cardiac death and myocardial infarction were considered "hard" events, and coronary angioplasty and bypass surgery > 60 days after testing were considered "soft" events. RESULTS: Of the 1,702 patients studied, 1,131 had normal or equivocal scan results. A total of 10 events occurred in this group (1 cardiac death and 1 myocardial infarction [0.2% hard events]; 4 coronary angioplasty and 4 bypass surgery procedures [0.7% soft events]). The rates of hard events and referral to catheterization after SPECT were similarly low in patients with a low (< 0.15), intermediate (0.15 to 0.85) and high (> 0.85) post-exercise treadmill test (ETT) likelihood of coronary artery disease. With respect to scan type, patients with normal, probably normal or equivocal scan results had similarly low hard event rates. In the 571 patients with abnormal scan results, there were 43 hard events (7.5%) and 42 soft events (7.4%) (p < 0.001 vs. 1,131 patients with normal scan results for both). When the complete spectrum of scan responses was considered, SPECT provided incremental prognostic value in all patient subgroups analyzed. However, the nuclear scan was cost-effective only in patients with interpretable exercise ECG responses and an intermediate to high post-ETT likelihood of coronary artery disease and in those with uninterpretable exercise ECG responses and an intermediate to high pre-ETT likelihood of coronary artery disease. CONCLUSIONS: Normal or equivocal exercise Tc-99m sestamibi study results are associated with a benign prognosis, even in patients with a high likelihood of coronary artery disease. Although incremental prognostic value is added by nuclear testing in all patient subgroups, a testing strategy incorporating nuclear testing proved to be cost-effective only in the groups with an intermediate to high likelihood of coronary artery disease before scanning.     

Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.     

Mammary gland expression of transgenes and the potential for altering the properties of milk

Transgenic animals are a useful in vivo experimental model for assessing the ability and impact of foreign gene expression in a biological system. Transgenic mice are most commonly used, while transgenic sheep, goats, pigs and cows have also been developed for specific, "applied" purposes. Most of the work directed at targeting expression of transgenes to the mammary gland of an animal, by using a milk gene promoter, has been with the intent of either studying promoter function or recovering the desired protein from the milk. Transgenic technology can also be used to alter the functional and physical properties of milk resulting in novel manufacturing properties. The properties of milk have been altered by adding a new protein with the aim of improving the milk, not of recovering the protein for other uses.     

Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis

The mechanism by which glucose stimulates insulin secretion from the pancreatic islets of Langerhans is incompletely understood. It has been suggested that malonyl-CoA plays a regulatory role by inhibiting fatty acid oxidation and promoting accumulation of cytosolic long-chain acyl-CoA (LC-CoA). In the current study, we have re-evaluated this "long-chain acyl-CoA hypothesis" by using molecular and pharmacologic methods to perturb lipid metabolism in INS-1 insulinoma cells or rat islets during glucose stimulation. First, we constructed a recombinant adenovirus containing the cDNA encoding malonyl-CoA decarboxylase (AdCMV-MCD), an enzyme that decarboxylates malonyl-CoA to acetyl-CoA. INS-1 cells treated with AdCMV-MCD had dramatically lowered intracellular malonyl CoA levels compared with AdCMV-betaGal-treated cells at both 3 and 20 mM glucose. Further, at 20 mM glucose, AdCMV-MCD-treated cells were less effective at suppressing [1-14C]palmitate oxidation and incorporated 43% less labeled palmitate and 50% less labeled glucose into cellular lipids than either AdCMV-betaGAL-treated or untreated INS-1 cells. Despite the large metabolic changes caused by expression of MCD, insulin secretion in response to glucose was unaltered relative to controls. The alternative, pharmacologic approach for perturbing lipid metabolism was to use triacsin C to inhibit long-chain acyl-CoA synthetase. This agent caused potent attenuation of palmitate oxidation and glucose or palmitate incorporation into cellular lipids and also caused a 47% decrease in total LC-CoA. Despite this, the drug had no effect on glucose-stimulated insulin secretion in islets or INS-1 cells. We conclude that significant disruption of the link between glucose and lipid metabolism does not impair glucose-stimulated insulin secretion in pancreatic islets or INS-1 cells.