43-kD Protein Research Articles

The zinc finger protein Gfi1 acts upstream of TNF to attenuate endotoxin‐mediated inflammatory responses in the lung

Gfi1 is a 55-kD nuclear zinc finger protein that is differentially expressed in lymphoid and myeloid cells. Gfi1(-/-) mice show a very strong systemic response to the endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here we report that the pathohysiological processes for this exaggerated inflammatory response take place in the lung. After LPS treatment, lungs of Gfi1(-/-) mice showed a rapid accumulation of mononuclear cells and a significant overproduction of inflammatory cytokines such as TNF, IL-1beta and IL-6. Increased cytokine production was also observed in blood-free perfused lungs from Gfi1(-/-) mice exposed to either LPS or overventilation. Alveolar macrophages but not airway epithelial cells from Gfi1(-/-) mice were found to be responsible for the enhanced cytokine production. Strikingly, when the TNF gene was deleted, Gfi1(-/-) animals were completely rescued from LPS hypersensitivity and had significantly lower IL-1beta and IL-6 levels. We conclude that the unrestrained endotoxin response of Gfi1(-/-) mice occurs mainly in the lung and that Gfi1 represents a novel factor limiting the inflammatory immune response of this organ, and propose that Gfi1 exerts its regulatory function in alveolar macrophages downstream of the LPS receptor (TLR4) and upstream of TNF.

Abscisic acid stimulates a calcium-dependent protein kinase in grape berry.

It has been demonstrated that calcium plays a central role in mediating abscisic acid (ABA) signaling, but many of the Ca2+-binding sensory proteins as the components of the ABA-signaling pathway remain to be elucidated. Here we identified, characterized, and purified a 58-kD ABA-stimulated calcium-dependent protein kinase from the mesocarp of grape berries (Vitis vinifera x Vitis labrusca), designated ACPK1 (for ABA-stimulated calcium-dependent protein kinase1). ABA stimulates ACPK1 in a dose-dependent manner, and the ACPK1 expression and enzyme activities alter accordantly with the endogenous ABA concentrations during fruit development. The ABA-induced ACPK1 stimulation appears to be transient with a rapid effect in 15 min but also with a slow and steady state of induction after 60 min. ABA acts on ACPK1 indirectly and dependently on in vivo state of the tissues. Two inactive ABA isomers, (-)-2-cis, 4-trans-ABA and 2-trans, 4-trans-(+/-)-ABA, are ineffective for inducing ACPK1 stimulation, revealing that the ABA-induced effect is stereo specific to physiological active (+)-2-cis, 4-trans-ABA. The other phytohormones such as auxin indoleacetic acid, gibberellic acid, synthetic cytokinin N-benzyl-6-aminopurine, and brassinolide are also ineffective in this ACPK1 stimulation. Based on sequencing of the two-dimensional electrophoresis-purified ACPK1, we cloned the ACPK1 gene. The ACPK1 is expressed specifically in grape berry covering a fleshy portion and seeds, and in a developmental stage-dependent manner. We further showed that ACPK1 is localized in both plasma membranes and chloroplasts/plastids and positively regulates plasma membrane H+-ATPase in vitro, suggesting that ACPK1 may be involved in the ABA-signaling pathway.

Ral GTPases regulate neurite branching through GAP-43 and the exocyst complex

Neurite branching is essential for the establishment of appropriate neuronal connections during development and regeneration. We identify the small GTPase Ral as a mediator of neurite branching. Active Ral promotes neurite branching in cortical and sympathetic neurons, whereas Ral inhibition decreases laminin-induced branching. In addition, depletion of endogenous Ral by RNA interference decreases branching in cortical neurons. The two Ral isoforms, RalA and -B, promote branching through distinct pathways, involving the exocyst complex and phospholipase D, respectively. Finally, Ral-dependent branching is mediated by protein kinase C–dependent phosphorylation of 43-kD growth-associated protein, a crucial molecule involved in pathfinding, plasticity, and regeneration. These findings highlight an important role for Ral in the regulation of neuronal morphology.

Mycoplasma gallisepticum Hemagglutinin VlhA, Pyruvate Dehydrogenase PdhA, Lactate Dehydrogenase, and Elongation Factor Tu Share Epitopes with Mycoplasma imitans Homologues

Mycoplasma gallisepticum is a major pathogen of poultry. Mycoplasma imitans is genetically and antigenically closely related to M. gallisepticum, but so far, only a few proteins of M. imitans have been identified as sharing epitopes with M. gallisepticum. In this study, we identified three proteins of M. gallisepticum that share with M. imitans epitopes defined by monoclonal antibodies (MAbs). MAb 9D4 reacted with the 67-kD hemagglutinin V1hA (previously termed pMGA) of M. gallisepticum and with its continuously expressed 40-kD protein. This MAb also reacted with a 40-kD protein of M. imitans, but not with its putative V1hA. Two-dimensional (2D) immunoblots of M. gallisepticum strains showed that their 40-kD proteins reacting with MAb 9D4 are expressed as major forms with isoelectric points (pI) around 6, and also as less-abundant forms differing in pI. In M. imitans, major forms of 40-kD proteins recognized by MAb 9D4 had pI around 6, whereas minor forms had pI between 5.5 and 5.8. The N-terminal sequence of the M. gallisepticum 40-kD protein recognized by MAb 9D4 strongly indicates that this protein is pyruvate dehydrogenase E1, subunit alpha (PdhA protein, also termed AcoA). The position of elongation factor Tu (EF-Tu), detected by the reference MAb GB8, was very similar in the 2D proteome maps of M. gallisepticum and M. imitans (MW of about 45 kD; pI - 5.6). In both M. gallisepticum and M. imitans, MAb 7G1 reacted with proteins of about 36 kD with similar charges (major forms with pI of about 8). The position of this protein in the proteome map of M. gallisepticum and its N-terminal sequence strongly suggest that MAb 7G1 recognizes lactate (malate) dehydrogenase (Ldh or Mdh). Comparison of 2D proteomes of 10 M. gallisepticum strains indicated that positions of EF-Tu, PdhA, and Ldh proteins are rather consistent and can be used as reference points in further analyses of the M. gallisepticum proteome.

Differential expression of the 67 kilodalton laminin receptor in epithelioid malignant mesothelioma and carcinomas that spread to serosal cavities

Expression of the 67-kd laminin receptor (67-kd LR) has been reported in a wide range of carcinomas, in many of which it correlated with poor differentiation, metastasis, disease progression, and poor survival. Malignant mesothelioma (MM) is a locally aggressive and highly lethal tumor of serosal cavities that is rarely associated with clinically detectable metastasis to distant organs. The aim of this study was to analyze the expression of the 67-kd LR in epithelioid MM and carcinomas in effusions. Effusions from patients diagnosed with ovarian (=24) and breast (=38) adenocarcinomas and MM (=24) (total = 86) were analyzed for 67-kd LR protein expression, using immunocytochemistry. The 67-kd LR mRNA expression was additionally analyzed in 21 MM effusions using reverse transcription-polymerase chain reaction (RT-PCR). Protein expression of the 67-kd LR was frequently detected in carcinomas (19/24 ovarian tumors, 79%; 15/38 breast tumors, 39%), but was rare in MM (2/24 cases, 8%), despite the presence of mRNA transcripts for the receptor in all 21 specimens studied using RT-PCR. Nine benign effusions that were additionally studied for protein expression were uniformly negative, as were all reactive mesothelial cells in malignant effusions. Our results suggest that the 67-kd LR may aid in the differential diagnosis between metastatic carcinoma, mainly of ovarian origin, and MM. They additionally suggest that the failure of MM to express the 67-kd LR protein, as opposed to the frequent expression in carcinomas with proven metastatic capacity, may be one of the factors contributing to the reduced ability of the former tumor to metastasize to distant organs.

External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice

During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins.

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.

Preventive and curative role of a 43kD protein from the leaves of the herb L on thioacetamide-induced hepatotoxicity in vivo

An approximately 43kD protein has been isolated and purified from the herb Cajanus indicus L and believed to be the most active principle for its hepatoprotective action. In this study, experiments have been performed to evaluate the effectiveness of that protein for the preventive and curative action against thioacetamide-induced toxicity in vivo using a murine model. Mice were treated with the protein intraperitoneally at a dose of 2mg/kg body weight for 2 and 6 days before and separately 1-5 days after thioacetamide administration to evaluate its preventive and curative role, respectively. Thioacetamide was administered once at a dose of 150mg/kg body weight and after 48h of its application, the animals were sacrificed. Levels of various markers related to physiological and pathological conditions of the liver, e.g., glutamate pyruvate transaminase (GPT), alkaline phosphatase (ALP), etc. were determined in the murine sera under different experimental conditions. In addition, antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) as well as thiobarbituric acid reactive substances (TBARS), were measured from the liver homogenates. The antioxidant property of the protein was compared with the potent antioxidant, vitamin E (used as a positive control). The active principle effectively reduced the elevated GPT and ALP levels in serum and lipid peroxidation in the liver tissue. The reduced levels of SOD, CAT and GST by thioacetamide were again brought back to almost normal levels upon pre- and post-treatment with the protein. Histopathological changes in the liver of TAA control and protein-treated groups also prove that the protein possesses hepatoprotective activity. The protein acts dose-dependently and maximum hepatoprotectivity was obtained when administered at a dose of 2mg/kg body weight. Data suggest that the active principle plays an important preventive and curative role against thioacetamide-induced hepatotoxicity.

Spatio‐temporal regulation and cleavage by matrix metalloproteinase stromelysin‐3 implicate a role for laminin receptor in intestinal remodeling during Xenopus laevis metamorphosis

The 37-kd laminin receptor precursor (LR) was first identified as a 67-kd protein that binds laminin with high affinity. We have recently isolated the Xenopus laevis LR as an in vitro substrate of matrix metalloproteinase stromelysin-3 (ST3), which is highly upregulated during intestinal metamorphosis in Xenopus laevis. Here, we show that LR is expressed in the intestinal epithelium of premetamorphic tadpoles. During intestinal metamorphosis, LR is downregulated in the apoptotic epithelium and concurrently upregulated in the connective tissue but with little expression in the developing adult epithelium. Toward the end of metamorphosis, as adult epithelial cells differentiate, they begin to express LR. Furthermore, LR is cleaved during intestinal remodeling when ST3 is highly expressed or in premetamorphic intestine of transgenic tadpoles overexpressing ST3. These results suggest that LR plays a role in cell fate determination and tissue morphogenesis, in part through its cleavage by ST3. Interestingly, high levels of LR are known to be expressed in tumor cells, which are often surrounded by fibroblasts expressing ST3, suggesting that LR cleavage by ST3 plays a role in both physiological and pathological processes.

Aggravation of Anti-Myeloperoxidase Antibody-Induced Glomerulonephritis by Bacterial Lipopolysaccharide: Role of Tumor Necrosis Factor-α

Aggravation of Anti-Myeloperoxidase Antibody-Induced Glomerulonephritis by Bacterial Lipopolysaccharide: Role of Tumor Necrosis Factor-α

Cytokine-Induced Endothelial Arginase Expression Is Dependent on Epidermal Growth Factor Receptor

L-arginine is metabolized to nitric oxide (NO) by NO synthase (NOS), or to urea and L-ornithine by arginase. L-ornithine contributes to vascular remodeling in pulmonary hypertension via metabolism to polyamines and proline. Previously we found that cytokines upregulate both NOS and arginase in pulmonary arterial endothelial cells. We hypothesized that cytokine-induced arginase I and II expression depend on epidermal growth factor (EGF) receptor (EGFR) activity. Bovine pulmonary arterial endothelial cells were treated with lipopolysaccharide and tumor necrosis factor-alpha (L/T). L/T treatment resulted in a substantial increase in urea production, and this increase in urea production was potently inhibited by both genistein and AG1478, inhibitors of EGFR. Levels of arginase I protein and arginase II mRNA were increased in response to L/T treatment, and genistein prevented the L/T-induced elevations in both arginase I protein and arginase II mRNA levels. L/T treatment increased production of nitrites and inducible NOS mRNA accumulation, and genistein and AG1478 had little effect on these changes. EGF (50 ng/ml) treatment resulted in enhanced urea production. Finally, a 170-kD protein was phosphorylated upon treatment with either EGF or L/T. Our results indicate that arginase induction by L/T depends in part on EGFR activity. We speculate that EGFR inhibitors may attenuate vascular remodeling without affecting NO release, and thus may represent novel therapeutic modalities for pulmonary hypertensive disorders.

Identification of Tyrosine Residues on ELMO1 That Are Phosphorylated by the Src-Family Kinase Hck

The SH3 and SH2 domains of hematopoietic cell kinase (Hck) play important roles in substrate targeting. To identify new components of Hck signaling pathways, we identified proteins that bind to the SH3 domain of Hck (Scott et al. (2002) J. Biol. Chem. 277, 28238). One such protein was ELMO1, the mammalian orthologue of the Caenorhabditis elegans gene, ced-12. ELMO1 is an approximately 80-kD protein containing a PH domain and a C-terminal Pro-rich sequence. In C. elegans, ced-12 is required for the engulfment of dying cells and for cell migration. In mammalian fibroblasts, ELMO1 binds to Dock180, and functions upstream of Rac during phagocytosis and cell migration. We previously showed that ELMO1 binds directly to the Hck SH3 domain and is phosphorylated by Hck. In this study, we used mass spectrometry to identify the following sites of ELMO1 phosphorylation: Tyr 18, Tyr 216, Tyr 511, Tyr 395, and Tyr 720. Mutant forms of ELMO1 lacking these sites were defective in their ability to promote phagocytosis and migration in fibroblasts. Single tyrosine mutations showed that Tyr 511 is particularly important in mediating these biological effects. These mutants displayed comparable binding to Dock180 and Crk as wild-type ELMO1, but gave a lowered activation of Rac. The data suggest that Src family kinase mediated tyrosine phosphorylation of ELMO1 might represent an important regulatory mechanism that controls signaling through the ELMO1/Crk/Dock180 pathway.

150-kD Oxygen-regulated Protein Is an Essential Factor for Insulin Release

This study was designed to show the induction and function of 150-kD oxygen-regulated protein (ORP150) in insulin secretion in vitro. A mouse beta-cell line, MIN6 cells, was cultured in medium containing potassium channel openers or various concentrations of glucose (2-25 mmol/L). ORP150 expression was studied by reporter assay, Western blot, or Northern blot analysis, concomitantly with insulin secretion. In addition, MIN6 cells infected with ORP150 recombinant adenovirus were adopted to show the function of ORP150 in insulin release. ORP150 expression in MIN6 cells was suppressed dose-dependently by the potassium channel opener diazoxide. Both low glucose (<2 mmol/L) and high glucose concentrations (25 mmol/L glucose) significantly induced more ORP150 expression compared with 10 mmol/L glucose. The treatment with diazoxide or infection of ORP150 antisense adenovirus suppressed ORP150 expression, and glucose-stimulated insulin secretion was effectively prevented. These findings show the involvement of ORP150 in insulin secretion in MIN6 cells.

Controlling the Gap

See related article, pages 558–566 Effective myocardial contraction depends in part on the efficient propagation of electrical signals through the tissue. The principal structure allowing for such rapid myocardial depolarization is the gap junction plaque, a collection of intercellular pores located primarily at intercalated discs. There, at myocyte poles, gap junctions function in close proximity to the complexes responsible for cell–cell tethering—desmosomes and fascia adherens junctions. Thus, the intercalated disc is the site of both intercellular coupling and adhesion. Gap junctions are formed by clusters of paired hexamers, one from each cell, which bridge to form a low-resistance intercellular channel facilitating the transmission of ions and small molecules (<1000 kD). Each hexamer (or connexon) is comprised of six individual connexins, a diverse protein family found in vascular, neural, and cardiac tissue. In humans, ventricular gap junctions are formed primarily from connexin43 (Cx43), a 43-kD protein encoded by chromosome 6.1 Cx43 is a dynamic molecule, with rapid turnover (t1/2 roughly 1 hour)2 and responsiveness to a variety of mechanical and chemical stimuli. Myocyte connectivity, as mediated by gap junctions, depends not only on net expression of Cx43, but on effective translocation of Cx43 to the cell surface, the phosphorylation state of both Cx43 and associated proteins,3 and intracellular pH.4 Ventricular Cx43 net protein expression is downregulated in various cardiac disease conditions such as end-stage ischemic,5 idiopathic,5 hypertrophic,1,6 and tachycardia-induced induced cardiomyopathies,7 myocardial ischemia and hibernation,8 and allograft rejection.9 …

A Kinase-Free BDNF Signaling Mechanism

Brain-derived neurotrophic factor (BDNF) signals through TrkB receptors to affect neuronal survival and differentiation as well as synaptic plasticity. Although full-length TrkB acts as a receptor tyrosine kinase, two truncated isoforms with distinct C termini that lack kinase activity have also been described that have been hypothesized to act as endogenous dominant negatives that inhibit signaling through the full-length isoform; alternatively, they may signal independently of it. Ohira et al. used the C-terminal sequence of one of these truncated isoforms (T1) to affinity purify a 28-kD T1-binding protein from the cytosolic fraction of adult rat brains, which they identified as Rho GDP dissociation inhibitor 1 (GDI1). GDI1, which binds Rho guanosine triphosphatases and inhibits GDP dissociation (thereby stabilizing their inactive form), bound T1 in an in vitro assay and coimmunoprecipitated with T1 that was transfected into HEK293 cells. Of the known receptors for BDNF, only T1 was expressed in long-term primary astrocyte cultures from neonatal rat hippocampus or cortical astrocytes in the adult rat brain. BDNF promoted T1 dissociation from GDI1 in long-term cultures of astrocytes; inhibited the activity of the GDI1 substrates RhoA, Cdc42, and Rac1; and elicited a change in astrocyte morphology from a fibrous to a flattened shape. Transfection of T1 or a deletion mutant into short-term astrocyte cultures (which did not express endogenous T1) confirmed that the effects of BDNF on Rho GTPase activity and morphology depended on T1. Thus, BDNF can signal directly through T1 to affect Rho GTPase activity and thereby astrocyte morphology. K. Ohira, H. Kumanogoh, Y. Sahara, K. J. Homma, H. Hirai, S. Nakamura, M. Hayashi, A truncated tropo-myosine-related kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1. J. Neurosci. 25 , 1343-1353 (2005). [Abstract] [Full Text]

Pemphigoid Nodularis: Two Case Studies and Analysis of Autoantibodies before and after the Development of Generalized Blistering

We report 45- and 61-year-old women with generalized prurigo nodularis-like eruption whose clinical, histologic and immunopathologic features were consistent with the diagnosis of pemphigoid nodularis. In one case, nodular lesions preceded the onset of generalized blistering by two years and in the other, no definite blister nor erosion was seen except for some appearing on the soles during the course of the disease. Western immunoblotting of EDTA-separated epidermal extracts revealed that the 230-kD bullous pemphigoid (BP) antigen was recognized by circulating autoantibodies in the patient sera, but the 180-kD BP antigen was not. The 180-kD BP antigen was recognized weakly by immunoblotting of the 180-kD BP antigen NC16a domain fusion protein, which shows high detection sensitivity. These findings suggested that weak reactivity of autoantibodies with either whole or a part of the 180-kD BP antigen molecule in some way accounts for negligible or localized blister-formation in this disorder. However, no particular change was noted in the reactivity with 180-kD BP antigen between the patient serum obtained before and after the development of generalized blistering. It is possible that different factors from the changes in serum reactivity with BP antigens may be involved in initiating generalized blistering.

Preparation of monoclonal antibodies against SARS coronavirus and staining usage in lung autopsy specimens

To prepare monoclonal antibodies against SARS coronavirus (SARS-CoV) on the purpose to explore the diagnosis methods of SARS. Female BALB/C mice were immunized with disinfected SARS-CoV (PUMC01) and the spleen cells were fused with myeloma NS-1 cells. The hybridoma cell strains were screened by enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA) and Western blotting. Autopsy lung tissue sections from SARS patients were stained with ascites of monoclonal antibody (M2 strain) by immunohistochemical technique. Six strains of hybridomas that produced the monoclonal antibodies against SARS-CoV were obtained. The hybridomas were tested to have specific reactions with SARS-CoV and no cross-reactivates with other common respiratory disease causing pathogens. Of the 6 strains, 1 was identified as the immunoglobulin G3 (IgG3) isotype, 5 were IgG1. Western blotting showed that one strain (M2) reacted with 68000- Dalton (68KD) protein and four strains with 27000-dalton (27KD) protein. Band of M4 stain was not got by western blotting. Brown particles were seen in macrophages and pneumocytes in autopsy lung tissues from SARS patients. Monoclonal antibodies we made were specific to the SARS-CoV, and they had been used to detect SARS-CoV in autopsy lung tissues specimens with positive results.

Reconstituting Modular Activity from Separated Domains of 6-Deoxyerythronolide B Synthase

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.

The 150-Kilodalton Oxygen-Regulated Protein Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice

The 150-kd oxygen-regulated protein is a novel stress protein that is located in the endoplasmic reticulum and contributes to cell survival when this organelle is under stress. Expression of this protein was strongly increased in alveolar macrophages and alveolar epithelial cells from mice with acute lung injury induced by lipopolysaccharide. Transgenic mice overexpressing the 150-kd protein showed decreased histological severity of this lung injury, accompanied by lower total protein concentrations, and lactate dehydrogenase activity in bronchoalveolar lavage fluid. As indicated by nick end-labeling, lipopolysaccharide induced apoptosis in fewer alveolar wall cells in transgenic than in wild-type mice. Transgenic mice also showed increased survival after lipopolysaccharide injection (a log-rank test). Thus, the 150-kd protein, an endoplasmic reticulum-related molecular chaperone, is pivotal in resisting acute lung injury from lipopolysaccharide.

PORCINE ANTIBODY RESPONSES TO TAENIA SOLIUM ANTIGENS RGP50 AND STS18VAR1

Cysticercosis, a disease caused by the larval form of Taenia solium, is diagnosed by detection of specific antibodies or by imaging techniques. Our preferred immunologic assay for cysticercosis is the enzyme-linked immunoelectrodifusion transfer blot, or immunoblot, using the lentil lectin bound antigens from larval cysts. Antibody reactivity with any one of seven glycoproteins is diagnostic for cysticercosis. To develop a simple antibody detection assay for field use, we have synthesized an 8-kD diagnostic antigen, sTs18var1 (a secreted protein with a mature size of 67 amino acids), and expressed a 50-kD membrane protein antigen, rGp50. We used these two diagnostic proteins in a quantitative Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) to measure the antibody responses in Peruvian pigs with cysticercosis. Three study designs were used. First, we followed the kinetics of antibody responses against these two diagnostic proteins in pigs with cysticercosis that were treated with oxfendazole. Second, we measured antibody response in naive experimentally infected pigs. Third, we followed the maternal antibodies against rGp50 and sTs18var1 in piglets born from sows with cysticercosis. These studies showed that antibody responses against the two diagnostic proteins in the FAST-ELISA are quantitatively correlated with infection by viable cysts, with anti-sTs18var1 activity being most responsive to the status of infection.