Predicting specificity in bZIP coiled-coil protein interactions

We present a method for predicting protein-protein interactions mediated by the coiled-coil motif. When tested on interactions between nearly all human and yeast bZIP proteins, our method identifies 70% of strong interactions while maintaining that 92% of predictions are correct. Furthermore, cross-validation testing shows that including the bZIP experimental data significantly improves performance. Our method can be used to predict bZIP interactions in other genomes and is a promising approach for predicting coiled-coil interactions more generally.     

Comparative immunocytochemical localization of lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) proteins: changes in the expression of LOXL during development and growth of mouse tissues

Lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are extracellular enzymes that deaminate peptidyl lysyl residues involved in the cross-linking of fibrillar collagens and elastin. While LOX is required for the survival of newborn mice, the role of LOXL during development remains unclear. Studies have shown that the same cell types express LOX and LOXL in the same tissues, but no functional differences have been established. We have compared the immunohistochemical localization of LOX and LOXL in various tissues from normal, young adult mice. LOX and LOXL were co-localized in the skin, aorta, heart, lung, liver and cartilage, but were localized to different areas in the kidney, stomach, small intestine, colon, retina, ovary, testis and brain. LOXL expression was further examined in tissues from different developmental stages. In embryonic mice (10.5–14.5 dpc), LOXL immunostaining was abundant in the heart, liver, intestine, and neural tube. LOXL was present in most major organs in late fetal (16.5 dpc) and newborn mice, but generally diminished as animals aged. Immunoreactivity was significantly reduced in the heart, lung, kidney and liver of 2 year-old mice, but remained prevalent in the skin and tongue. LOX and LOXL were also found in the nuclei of cells in a number of tissues. These results indicate that LOXL has a role during mouse development and in the maintenance of adult tissues.     

High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein

Oxidatively modified low-density lipoprotein (LDL), generated as a result of incubation of LDL with specific cells (e.g., endothelial cells, EC) or redox metals like copper, has been suggested to be an atherogenic form of LDL. Epidemiological evidence suggests that higher concentrations of plasma high-density lipoprotein (HDL) are protective against the disease. The effect of HDL on the generation of the oxidatively modified LDL is described in the current study. Incubation of HDL with endothelial cells, or with copper, produced much lower amounts of thiobarbituric acid-reactive products (TBARS) as compared to incubations that contained LDL at equal protein concentrations. Such incubations also did not result in an enhanced degradation of the incubated HDL by macrophages in contrast to similarly incubated LDL. On the other hand, inclusion of HDL in the incubations that contained labeled LDL had a profound inhibitory effect on the subsequent degradation of the incubated LDL by the macrophages while having no effect on the generation of TBARS or the formation of conjugated dienes. This inhibition was not due to the modification of HDL as suggested by the following findings. (A) There was no enhanced macrophage degradation of the HDL incubated with EC or copper alone, together with LDL, despite an increased generation of TBARS. (B) HDL with the lysine groups blocked (acetyl HDL, malondialdehyde (MDA) HDL) was still able to prevent the modification of LDL and (C) acetyl HDL and MDA-HDL competed poorly for the degradation of oxidatively modified LDL. It is suggested that HDL may play a protective role in atherogenesis by preventing the generation of an oxidatively modified LDL. The mechanism of action of HDL may involve exchange of lipid peroxidation products between the lipoproteins.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.     

Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis

TNF and IFN-gamma are thought to be involved in the immune response to mycobacterial infection because they exhibit antimycobacterial effects in vitro. To investigate the roles of these cytokines in vivo at the site of disease activity in human tuberculosis, we evaluated local cytokine production in patients with tuberculous pleuritis. Both TNF and IFN-gamma were selectively concentrated 5- to 30-fold in pleural fluid, compared to blood of the same patients. Messenger RNA for both cytokines was detected in pleural tissue by in situ hybridization, suggesting that selective cytokine concentration is due to local cytokine production. Two Mycobacterium tuberculosis cell wall components, the protein-peptidoglycan complex and lipoarabinomannan, caused dose-dependent release of TNF by pleural fluid mononuclear cells and may constitute the stimuli for TNF production in the pleural space. In contrast to results obtained for TNF release, the protein-peptidoglycan complex, but not lipoarabinomannan, stimulated IFN-gamma release by pleural fluid mononuclear cells. The clinical manifestations of tuberculous pleuritis, such as fever, exudative pleural effusion, and tissue necrosis, may be due to the effects of elevated local TNF concentrations, produced in response to mycobacterial cell wall components.     

cDNA sequence and heterologous expression of the human neurokinin-3 receptor.

Functional cDNA clones encoding the human neurokinin-3 receptor were isolated from human brain mRNA. The cloned human neurokinin-3 receptor was expressed in COS cells and Xenopus oocytes, where peptide binding affinity and intracellular effector activation were determined. Neurokinin B is the most potent agonist, followed by eledoisin, substance K and substance P. The binding affinities of these peptides at the human neurokinin-3 receptor differ quantitatively from the rat receptor, implying a functional consequence of the sequence divergence between the two species. Heterologous expression in oocytes revealed that, unlike the neurokinin-1 receptor, the efficacy of ion channel activation mediated by the neurokinin-3 receptor does not approximate the binding affinity. The heterologous expression of the human neurokinin-3 receptor will facilitate further investigation into its biochemical functions.     

Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis.

Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.