Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

In vivo administration with IL-1 accelerates the development of collagen-induced arthritis in mice

In an attempt to examine the in vivo proinflammatory properties of IL-1, the effects of rIL-1 beta on the development of collagen-induced arthritis in mice were investigated. The results presented in this paper demonstrated that the administration of rIL-1 beta via mini-osmotic pumps into DBA/1 mice which were suboptimally immunized with native chick type II collagen (NcII) markedly accelerated the onset as well as the progression of the arthritic disease. When IL-1-containing osmotic pumps were s.c. implanted onto mice 18 days post-collagen immunization, clinical signs of arthritis appeared within 3 to 4 days after the implant with the pumps. Maximal incidence of arthritis which was usually 80 to 100% occurred between the 6th and 7th day after the administration of rIL-1 beta. Histologic analyses revealed that the knee and ankle joints from mice which were treated with rIL-1 beta for 7 days were most severely and consistently affected. Furthermore, these IL-1-treated mice exhibited granulocytic hyperplasia within the marrow as well as marked peripheral blood neutrophilia. By contrast, arthritis was not observed during the 7-day course of the IL-1 study in the following control groups: 1) mice that were only immunized with NcII, and 2) collagen-immunized mice which received osmotic pumps containing PBS. A substantial number of these collagen-immunized mice which were not treated with IL-1 eventually developed arthritis but at later times after the incidence of arthritis had peaked in the IL-1-treated group. In addition, unimmunized mice failed to develop arthritis upon treatments with IL-1 beta. Moreover, the humoral responses to NcII were not altered in the IL-1-treated mice. Thus, these in vivo studies suggest that IL-1 is potentially capable of triggering the various inflammatory events of collagen-induced arthritis, and thereby, contribute to the pathogenesis of murine arthritis.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Signaling of Ankle Joint Position by Receptors in Different Muscles

Plots were made of multiunit activity versus ankle joint position for receptors in each of the 12 muscles crossing the cat ankle joint, except peroneus tertius, by recording from populations of afferent fibers in muscle nerves. The discharge was measured 15 or 30 sec after terminating the movements that altered the position of the joint. These recordings were dominated by large-spike activity that would be expected to originate mainly from primary spindle endings. Seven of the 12 muscles also cross other joints. Their responses at a given ankle joint position were so altered by changes in the position of the knee or toe joints that they could not reliably signal the position of the ankle joint. As judged from multiunit recording, receptors in each of the five muscles specific to the ankle joint were influenced by more than one axis of ankle joint displacement.

Single-unit recording from dorsal root filaments was used to determine whether primary or secondary spindle receptors in soleus and tibialis anterior could selectively signal one axis of ankle joint rotation. Individual soleus receptors were tested both on the flexion extension axis and with a combined adduction–eversion movement.

For 38 of the 70 soleus receptors examined (54%), firm adduction–eversion produced a level of activity greater than that caused by 10° of flexion, and for 77% the level of activity was greater than that caused by 5° of flexion. For 168 of the 184 tibialis anterior receptors studied (91%), firm abduction inversion produced a level of activity greater than that caused by 10° of extension. Thus few receptors were found that responded exclusively to one axis of rotation.

One way in which the position of the ankle joint could be specified in the face of multiaxial receptor activity is by examining the receptor discharge from more than one muscle. A suggestion for how the nervous system might do this is given in the discussion.     

The glucose‐deprivation network counteracts lapatinib‐induced toxicity in resistant ErbB2‐positive breast cancer cells

Dynamic interactions between intracellular networks regulate cellular homeostasis and responses to perturbations. Targeted therapy is aimed at perturbing oncogene addiction pathways in cancer, however, development of acquired resistance to these drugs is a significant clinical problem. A network‐based computational analysis of global gene expression data from matched sensitive and acquired drug‐resistant cells to lapatinib, an EGFR/ErbB2 inhibitor, revealed an increased expression of the glucose deprivation response network, including glucagon signaling, glucose uptake, gluconeogenesis and unfolded protein response in the resistant cells. Importantly, the glucose deprivation response markers correlated significantly with high clinical relapse rates in ErbB2‐positive breast cancer patients. Further, forcing drug‐sensitive cells into glucose deprivation rendered them more resistant to lapatinib. Using a chemical genomics bioinformatics mining of the CMAP database, we identified drugs that specifically target the glucose deprivation response networks to overcome the resistant phenotype and reduced survival of resistant cells. This study implicates the chronic activation of cellular compensatory networks in response to targeted therapy and suggests novel combinations targeting signaling and metabolic networks in tumors with acquired resistance.     

Frequency and space representation in the primary auditory cortex of the frequency modulating batEptesicus fuscus

1. Frequency and space representation in the auditory cortex of the big brown bat, Eptesicus fuscus, were studied by recording responses of 223 neurons to acoustic stimuli presented in the bat's frontal auditory space. 2. The majority of the auditory cortical neurons were recorded at a depth of less than 500 microns with a response latency between 8 and 20 ms. They generally discharged phasically and had nonmonotonic intensity-rate functions. The minimum threshold, (MT) of these neurons was between 8 and 82 dB sound pressure level (SPL). Half of the cortical neurons showed spontaneous activity. All 55 threshold curves are V-shaped and can be described as broad, intermediate, or narrow. 3. Auditory cortical neurons are tonotopically organized along the anteroposterior axis of the auditory cortex. High-frequency-sensitive neurons are located anteriorly and low-frequency-sensitive neurons posteriorly. An overwhelming majority of neurons were sensitive to a frequency range between 30 and 75 kHz. 4. When a sound was delivered from the response center of a neuron on the bat's frontal auditory space, the neuron had its lowest MT. When the stimulus amplitude was increased above the MT, the neuron responded to sound delivered within a defined spatial area. The response center was not always at the geometric center of the spatial response area. The latter also expanded with stimulus amplitude. High-frequency-sensitive neurons tended to have smaller spatial response areas than low-frequency-sensitive neurons. 5. Response centers of all 223 neurons were located between 0 degrees and 50 degrees in azimuth, 2 degrees up and 25 degrees down in elevation of the contralateral frontal auditory space. Response centers of auditory cortical neurons tended to move toward the midline and slightly downward with increasing best frequency. 6. Auditory space representation appears to be systematically arranged according to the tonotopic axis of the auditory cortex. Thus, the lateral space is represented posteriorly and the middle space anteriorly. Space representation, however, is less systematic in the vertical direction. 7. Auditory cortical neurons are columnarly organized. Thus, the BFs, MTs, threshold curves, azimuthal location of response centers, and auditory spatial response areas of neurons sequentially isolated from an orthogonal electrode penetration are similar.     

Multiple sampling for increasing the diagnostic sensitivity of nipple aspirate fluid for atypical cytology

OBJECTIVE: To determine if repeated collection of nipple aspirate fluid (NAF) can improve the diagnostic sensitivity for cytologic atypia, a marker of increased risk of breast cancer. STUDY DESIGN: Two hundred sixty-seven women without known breast disease volunteered for NAF cytology at 5 6-month intervals over 2 years. NAF samples were prepared on Millipore filters (Millipore Filter Corp., Bedford, Massachusetts, U.S.A.) and stained with a modified Papanicolaou method. Fluid availability and cellular abnormalities were evaluated for each collection attempt. Cellular findings were classified as benign, hyperplasia or atypia. RESULTS: NAF was obtained from 178 women (66.6%) at the first visit and from an additional 15, 10, 2 and 4 women at visits 2, 3, 4 and 5, respectively, for a cumulative total of 78.2% by visit 5. The number of women yielding NAF containing hyperplastic or atypical epithelial cells was determined at each visit. Hyperplastic cells were found in 34 (19.1%) at visit 1 and in an additional 20, 10, 5 and 4 women at visits 2, 3, 4 and 5, respectively. Atypical epithelial cells were present in 12 (6.7%) women at the initial visit and in an additional 11, 7, 5 and 1 women at visits 2, 3, 4 and 5, respectively, for a cumulative percent of 18.2 at visit 5. NAF could not be obtained from 58 women at any visit. CONCLUSION: These findings suggest that an optimum collection method for NAF cytology should consist of at least 3 or 4 separate fluid aspiration attempts. Reviewing repeated multiple samples instead of 1 increases the number of women who can be evaluated and the likelihood of detecting cytologic atypia.     

Evidence of Highly Conserved β-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens*

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H₂O₂, whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.Aging lens crystallins accumulate post-synthetic modifications that can be broadly classified into three categories, namely (1) protein backbone changes, such as racemization and truncation (1–3), (2) conversion of one amino acid into another, such as deamidation of asparagine into aspartate or deguanidination of arginine into ornithine, deamination of lysine into allysine and 2-aminoadipic acid (4–6), and (3) amino acid residue damage from reactive carbonyls and reactive oxygen species (7,8). Carbonyl damage results from the Maillard Reaction by glucose, methylglyoxal, or oxidation products of ascorbate, tryptophan or lipids which form adducts and crosslinks with nucleophilic group of lysine, arginine and cysteine. Examples include carboxymethyl-lysine, pentosidine, methylglyoxal hydroimidazolones, HNE-cysteine adducts and kynurenine (7,9–12). Oxidative damage results from reactive oxygen species that directly damage amino acid residues, e.g. oxidizing tryptophan into N-formyl kynurenine and kynurenine, methionine into its sulfoxide, and cysteine into cysteine disulfides or cysteic acid (13–15).Because of their relevance to age-related cataract, the impact of each of these modifications on crystallin structure and stability is the subject of intense investigation. Importantly, Benedek proposed that high molecular weight (HMW)¹ crystallin aggregates the size of 50 million daltons are needed in order for lens opacification to be visible(16,17). Crystallin aggregation conceivably occurs by one of several mechanisms that include conformational changes as a consequence amino acid mutations (18) or physical-chemical protein modifications. Of the latter, one mechanism that is dominant in several types of cataract involves oxidation of cysteines into protein disulfides (18) and formation of HMW aggregates that scatter light (19).In order to mimic the oxidative process and formation of protein disulfides linked to low concentrations of glutathione (GSH) in the nucleus of the human lens, we recently created the LEGSKO mouse in which lenticular GSH was lowered by knocking out the γ-glutamyl cysteine ligase subunit Gclc (20). These mice develop full-blown nuclear cataract by about 9 months and represent an important model for the development of drugs that might block or reverse the oxidation of crystallin sulfhydryls and presumably protein aggregation. However, this assumption in part depends on whether the sites of disulfide bond formation are similar in mouse and human age-related cataract. To test this hypothesis we performed the first comparative analysis of the cataract prone LEGSKO mouse and human aging and cataractous lens crystallin disulfidome, and compared the results with the disulfidome from mouse lens homogenate oxidized in vitro with H₂O₂ as a model of crystallin aggregation and opacification.     

Reaction mechanism of isomaltooligosaccharides synthesis by α-glucosidase fromAspergillus carbonarious

Summary An -glucosidase fromAspergillus carbonarious CCRC 30414 was employed for investigating the enzymatic synthesis of isomaltooligosaccharides from maltose. The enzyme transferred a glucose unit from the nonreducing end of maltose and other -linked glucosyl oligosaccharides to glucose and other glucosyl oligosaccharides which function as accepting co-substrates. The transfer of a glucose unit occurs most frequently to the 6-OH position of the nonreducing end of acceptor, but transfer to 4-OH position also occurs. Treatment of 30 % (w/v) maltose with the enzyme under optimum conditions afforded more than 50% isomaltooligosaccharides.     

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.