Crystal structure of a gamma-herpesvirus cyclin-cdk complex.

Several gamma-herpesviruses encode proteins related to the mammalian cyclins, regulatory subunits of cyclin-dependent kinases (cdks) essential for cell cycle progression. We report a 2.5 A crystal structure of a full-length oncogenic viral cyclin from gamma-herpesvirus 68 complexed with cdk2. The viral cyclin binds cdk2 with an orientation different from cyclin A and makes several novel interactions at the interface, yet it activates cdk2 by triggering conformational changes similar to cyclin A. Sequences within the viral cyclin N-terminus lock part of the cdk2 T-loop within the core of the complex. These sequences and others are conserved amongst the viral and cellular D-type cyclins, suggesting that this structure has wider implications for other cyclin-cdk complexes. The observed resistance of this viral cyclin-cdk complex to inhibition by the p27(KIP:) cdk inhibitor is explained by sequence and conformational variation in the cyclin rendering the p27(KIP:)-binding site on the cyclin subunit non-functional.

Monoclonal antibodies to CD45 modify LPS-induced arachidonic acid metabolism in macrophages.

Signaling by lipopolysaccharide (LPS) through CD14 involves the activation of protein tyrosine kinases of the src family and leads to cytokine production and activation of arachidonic acid metabolism in macrophages. CD45 protein tyrosine phosphatase (PTPase) might play a role in modulating the response through this pathway. Although a critical role in regulation of T-cell signaling for CD45 has been demonstrated, little is known about its role in macrophages. Monoclonal antibodies to CD45 and F(ab')(2) fragments of the monoclonal antibody enhanced the response of differentiated THP-1 monocytic cells to LPS for the release of radiolabeled arachidonic acid metabolites, prostaglandin E(2), and tumor necrosis factor alpha. The enhancing effect of anti-CD45 mAbs was shown to occur primarily through CD14-dependent signaling by performing the experiments under conditions favoring that pathway. Further, LPS may be able to alter the enzymatic activity of CD45, as shown by Western blots of CD45 immunoprecipitates in which LPS caused a transient change in the phosphorylation state of CD45. We conclude that CD45 appears to play a role in LPS-induced responses through the CD14 pathway, possibly through its PTPase activity.

Overcoming inhibitions: subversion of CKI function by viral cyclins.

DNA tumour viruses deregulate the mammalian cell cycle to provide a better environment for their replication. Studies of such deregulation have led to the identification of key regulatory steps that normally control the G1-S phase transition of the cell cycle. The balance between the activities of G1-specific cyclin-CDK complexes and their inhibitors is critical. Recent studies suggest that certain herpesviruses disrupt this balance: the viruses encode a cyclin that generates active complexes even in the presence of high inhibitor levels.

A comparison of the effects of lipopolysaccharide and ceramide on arachidonic acid metabolism in THP-1 monocytic cells.

Ceramide has been shown to be an important second messenger for signal transduction in cells of myeloid lineage. Studies have suggested that lipopolysaccharide (LPS) may activate signaling pathways by mimicking the action of ceramide. We explored this hypothesis with THP-1 cells in terms of the effects of LPS, C2 ceramide, and sphingomyelinase on arachidonic acid metabolism as measured by the release of radiolabeled eicosanoids. Arachidonic acid metabolism was activated by both LPS and ceramide. However, the ratio of prostaglandin E2 to leukotriene C4 was 10 times higher in cells treated with LPS than with ceramide. Unlike LPS, prior exposure to ceramide did not desensitize the cells to subsequent challenge with either LPS or ceramide, nor could LPS desensitize the cells to challenge with ceramide. The results suggest that, although LPS and ceramide may share signaling components, the signaling pathways are not identical.

Chromospheric Helium Imaging Photometer (an Instrument for High Time Cadence 1083-nm Wavelength Solar Observations).

The scientific motivation, design criteria, and specifications for a new ground-based instrument to observe the Sun in the He i 1083-nm spectral line is described. The instrument employs a liquid-crystal tunable Lyot-type spectral filter and an array detector that allows the full solar disk to be observed with a time cadence of minutes. We describe the telescope's optical and mechanical features and discuss computer interface and data-reduction procedures employed. Instrument performance during the initial year of operation of the telescope at its high-altitude site is summarized.

Activation of arachidonic acid metabolism in mouse macrophages by bacterial amphiphiles.

The relative activities of lipoteichoic acid (LTA) from four Gram-positive bacteria were compared to different lipopolysaccharide (LPS) preparations for activation of arachidonic acid metabolism in mouse peritoneal macrophages. Total eicosanoid was determined in cultures labeled with [3H]-arachidonic acid. Prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were determined by EIA analysis. The relative potencies of the different preparations were: smooth LPS from Salmonella abortus > or = Re-LPS from Salmonella minnesota (R-595) > or = LTA from Streptococcus pyogenes approximately Streptococcus faecalis approximately Staphylococcus aureus > or = monophosphoryl lipid A derived from the Re-LPS >> LTA from Bacillus subtilis. Activation of eicosanoid release was inhibited by staurosporin for all of the amphiphiles tested. Treatment of the macrophage cultures with LTA from S. pyogenes, S. faecalis, and S. aureus, either in the presence or absence of indomethacin, desensitized the cells to eicosanoid release on subsequent challenge with LPS. The desensitized cells remained responsive to the phorbol ester phorbol myristate acetate. LPS from Gram-negative bacteria has immunostimulatory and endotoxic activities which result, in part, from the release of eicosanoids and other mediators from activated macrophages. The similarities in the patterns of cell activation by LPS and LTA suggest that lipoteichoic acids might contribute to the pathogenicities of Gram-positive bacteria.

Carbonate inhibition of leukotriene D4-dipeptidase in human serum.

Human serum contains an inhibitor of leukotriene D4 (LTD4) dipeptidase which was separated from the enzyme by ultrafiltration (Amicon, YM-10). Removal of the inhibitor resulted in a 3- to 5-fold increase in total LTD4-dipeptidase activity in the material retained by the filter. Inhibitor activity (which was assayed with a partially purified LTD4-dipeptidase) was recovered in the filtrate. Ultrafiltration of serum using YM-3, YM-1, and YC-05 membranes suggested an inhibitor molecular weight of less than 500. Elution of inhibitor activity from a Bio Gel P2 gel filtration column was identical to the elution pattern of pure carbonate. The inhibitor was heat stable (95 degrees C, 30 min), stable in 0.1 N NaOH, but rapidly inactivated by 0.1 N HCl at both 4 degrees C and 30 degrees C. Partially purified LTD4-dipeptidase was inhibited by carbonate and phosphate but not by nitrate, sulfate, or chloride. Based on these observations it was concluded that the inhibitor of LTD4-dipeptidase in human serum either was carbonate or required carbonate. The relative concentrations of LTC4, LTD4, and LTE4 appear to be important parameters in determining the duration and intensity of LT mediated reactions. The relative concentration of carbonate in serum or extracellular fluids might, therefore, be a factor in modulating localized LT mediated responses.

Role of anionic lipid in bacterial membranes.

The major phospholipids of Bacillus stearothermophilus are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). Under the growth conditions used in this study the concentration of anionic lipid (PG + CL) was determined by the pH of the culture medium. Cells grown in a complex medium at pH 5.8, 7.0, and 8.0 contained 17, 29 and 36 nmol of anionic (PG + CL) lipid/mg cell (dry weight). The concentration of the zwitterionic lipid phosphatidylethanolamine (PE) was 17-20 nmol/mg cell (dry weight) under all conditions. Analysis of isolated membrane preparations suggested that the amount of anionic lipid per unit area of membrane increased as the pH of the growth medium was increased. Membranes from cells grown at pH 5.8 and 8.0 contained 130 and 320 nmol anionic lipid/mg membrane protein, respectively. Phosphatidylethanolamine appeared to be localized on the inner membrane surface in cells grown under all conditions. Increasing the ionic strength of the culture medium by the addition of NaCl or KCl had little effect on growth at pH 5.8 but inhibited growth at pH 7 and 8. It was concluded that anionic phospholipid plays an important physiological role in maintaining an acidic pH at the outer membrane surface.

Leukotriene metabolism and hypersensitivity in man and mouse.

The relative rates of cysteinyl-leukotriene metabolism were analyzed in fresh human and mouse serum. Human serum contained higher gamma-glutamyl-transpeptidase activity than mouse serum, and a higher percentage of the metabolized leukotriene C4 was recovered as leukotriene D4 in the human serum than in the mouse serum. The results suggest that the patterns of metabolism of the cysteinyl-leukotrienes could be an important factor in determining the relative sensitivity of an animal to the development of hypersensitivity reactions.

Products of phospholipid metabolism in Bacillus stearothermophilus.

The products of phospholipid turnover in Bacillus stearothermophilus were determined in cultures labeled to equilibrium and with short pulses of [32P]phosphate and [2-3H]glycerol. Label lost from the cellular lipid pool was recovered in three fractions: low-molecular-weight extracellular products, extracellular lipid, and lipoteichoic acid (LTA). The low-molecular-weight turnover products were released from the cells during the first 10 to 20 min of a 60-min chase period and appeared to be derived primarily from phosphatidylglycerol turnover. Phosphatidylethanolamine, which appeared to be synthesized in part from the phosphatidyl group of phosphatidylglycerol, was released from the cell but was not degraded. The major product of phospholipid turnover was LTA. Essentially all of the label lost from the lipid pool during the final 40 min of the chase period was recovered as extracellular LTA. The LTA appeared to be derived primarily from the turnover of cardiolipin and the phosphatidyl group of phosphatidylglycerol. Three types of LTA were isolated; an extracellular LTA was recovered from the culture medium, and two types of LTA were extracted from membrane preparations or whole-cell lysates by the hot phenol-water procedure. Cells contained 1.5 to 2.5 mg of cellular LTA per g of cells (dry weight), over 50% of which remained associated with the membrane when cells were fractionated. Over 75% of the 3H label incorporated into the cellular LTA pool during a 90-min labeling period was released from the cells during the first cell doubling after the chase. Label lost from the lipid pool was incorporated into cellular LTA which was then modified and released into the culture medium.

The relationships between growth temperature, fatty acid composition and the physical state and fluidity of membrane lipids in Yersinia enterocolitica.

The relationship between membrane lipid composition and membrane lipid phase transitions was investigated in Yersinia enterocolitica cells grown at 5, 22 and 37 degrees C. The total phospholipid concentrations were 9.4, 7.3 and 6.3% of the cell dry weight for cells grown at 5, 22 and 37 degrees C, respectively. The relative concentrations of the three major phospholipids, phosphatidylethanolamine (73--76%), phosphatidylglycerol (9--11%) and cardiolipin (11--13%) were essentially the same at all three growth temperatures. The ratios of unsaturated to saturated fatty acids were 2.2, 1.1 and 0.4 for cells grown at 5, 22 and 37 degrees C, respectively. This change in the fatty acid composition in response to temperature changes is similar to the patterns reported for other organisms. Reversible thermotropic phase transitions were detected by calorimetric analysis in both pure lipid preparations and membrane preparations. The mid-points of the thermotropic phase transitions were at --13, --9 and 1 degree C for membranes from cells grown at 5, 22 and 37 degrees C, respectively. The phase transitions of the membranes from cells grown at the three different temperatures occurred below the lowest growth temperature (5 degrees C). The alternations in the fatty acid composition in Y. enterocolitica did not, therefore, appear to be required to adjust membrane fluidity but might rather be required for some other membrane function.

Protein-associated lipid of Bacillus stearothermophilus.

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.

Effect of calcium and anaerobiosis on the thermostability of Bacillus stearothermophilus.

Bacillus stearothermophilus NCA 2184 lost viability and subsequently released cytoplasmic components when suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.2) and incubated at 60 degrees C. Cell lysis was prevented by the addition of 10 mM CaCl2 to the Tris-buffer suspension. Cells which were incubated under anaerobic conditions for 20 min in the growth medium before they were collected were stable in the Tris-buffer suspension without added calcium. Anaerobic incubation effected an increase in membrane cardiolipin which appeared to be related to the increase in the thermostability of the cells.

Metabolism of phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin of Bacillus stearothermophilus.

The total phospholipid content of Bacillus stearothermophilus was constant during exponential growth, increased during the transition from the exponential to stationary phase of growth, and then slowly increased during the stationary phase. The first increase was a result of an increase in phosphatidylethanolamine; the second was a result of an increase in cardiolipin. Cessation of aeration of an exponentially growing culture or suspension in a nongrowth medium resulted in an immediate reduction in the rate of total phospholipid and phosphatidylethanolamine synthesis and a quantitative conversion of phosphatidylglycerol to cardiolipin. Cardiolipin appeared to be synthesized by the direct conversion of two molecules of phosphatidylglycerol to cardiolipin. After a 20-min pulse of (32)P, phosphatidylglycerol showed the most rapid loss of (32)P followed by cardiolipin, whereas phosphatidylethanolamine did not lose (32)P. The loss of (32)P from the total lipid pool, phosphatidylglycerol, and cardiolipin was biphasic, with rapid loss during the first two bacterial doublings followed by a greatly reduced rate of loss. The major loss of (32)P from the total phospholipid pool appeared to be by breakdown of cardiolipin. The loss of (32)P from the lipid pool was energy dependent (i.e., did not occur under anaerobic conditions or in the absence of an energy source) and was dependent on some factor other than the concentration of cardiolipin in the cells. The apparent conversion of phosphatidylglycerol to cardiolipin was independent of energy metabolism. Chloramphenicol reduced the rate of turnover of both phosphatidylglycerol and cardiolipin. The rate of lipid synthesis (all phospholipid components) was constant for about 10 min after the addition of chloramphenicol but diminished markedly after 20 min. Turnover of (32)P incorporated into phospholipid during a 30-min period prior to the addition of chloramphenicol was more rapid after the removal of chloramphenicol than that of (32)P incorporated during a 30-min period in the presence of chloramphenicol.

Phospholipids from Bacillus stearothermophilus.

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.