Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor.

Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.

Biochemical analysis and crystallisation of Fc gamma RIIa, the low affinity receptor for IgG.

Fc gamma RIIa is one of a family of specific cell surface receptors for immunoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG isotypes, plays important roles in immune homeostasis. However, the precise characteristics of IgG binding and three-dimensional structure of Fc gamma RIIa have not been reported. This study describes the affinity of the Fc gamma RIIa:IgG interaction as well as biochemical characterisation of recombinant Fc gamma RIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 binds with an affinity of K(D) = 0.6 microM, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K(D) = 3 microM, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived Fc gamma RIIa established the genuine N-terminus with Q being the first amino acid in the sequence Q, A, A, A, P... extending the N-terminus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometry (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract X-rays beyond 2.1 A resolution in the space group P2(1)2(1)2 with cell dimensions a = 78.8 A, b = 100.5 A, c = 27.8 A. This marks a significant advance towards understanding the three-dimensional structure of Fc gamma RIIa and related FcR proteins that share high amino acid identity with Fc gamma RIIa.

Baculoviral expression of telomerase in primary human fibroblasts to rejuvenate cells for tissue engineering.

Tissue engineering involves the use of synthetic or natural materials as a scaffold to support the growth of replacement tissue or organs. The use of autologous cells to populate the scaffold avoids problems associated with rejection; however, a major limitation of this approach is the finite lifespan of primary cells in culture. This finite lifespan is due to the shortening of telomeres, short repetitive sequences of DNA located at the ends of eukaryotic chromosomes. Ectopic expression of telomerase reverse transcriptase (hTERT) is able to reconstitute telomerase activity and maintain the length of telomeres. This study investigated an alternative gene delivery vector, baculovirus, for the expression of hTERT in primary human cells. A recombinant baculovirus was used to efficiently deliver the hTERT gene to primary fibroblasts and the telomerase enzyme was found to be active. Although no increase in telomere length was detected, expression of hTERT in primary fibroblasts resulted in a significant extension of replicative lifespan. To our knowledge this is a novel attempt to use a recombinant baculovirus for the extension of cellular lifespan by exogenous expression of telomerase.

Nuclear medicine studies of aging--II. Femoral vessel uptake of 99mTc-diphosphonates vs age.

Bone images obtained after use of 99mTc-MDP were examined for activity presumed to be present in the femoral vessels. The criteria for this extraosseous uptake were: present on anterior views of the thighs, located medial to the femurs, extended outward and downward, approximately linear and continuous, and present on both thighs. Each image was graded as zero (not visible), one plus (barely detectable) and two plus (readily visualized). Using the mean age of each grouping (20-29 years depicted as age 25), the percent positive were noted to closely correlate with age. The earliest positive detected was in a 24 year old male; by age 80 years and greater, over 93% of the cases showed 1+ or 2+ activity. In addition, the average score increased linearly with age. These findings were correlated with literature data on femoral artery diameter and stiffness, and with information on aortic calcification.

In vitro analysis of pulmonary inflammation using rat lung organ cultures.

We have analyzed leukocyte to lung adhesive interactions and neutrophil-mediated lung injury using a rat lung organ culture system. Rat lung slices were maintained in tissue culture on gelatin sponges floating at the gas-liquid interface. Maintenance of three-dimensional alveolar structure, critical to the viability of lung tissue, was achieved by instilling 0.5% agarose (in 37 degrees C RPMI 1640) into the lungs during tissue explanation. Quantitative neutrophil to lung adhesive interactions were examined using an adaptation of the Woodruff-Stamper frozen section binding assay. Pretreatment of organ cultures with recombinant human tumor necrosis factor (rhTNF) resulted in a protein synthesis-dependent three- to fourfold increase in adhesiveness for neutrophils. Time course and mononuclear leukocyte binding experiments revealed that TNF-induced rat lung adhesiveness peaks at 4 h and is largely neutrophil-specific. Agonist-induced activation of neutrophils in the presence of [3H]leucine-labeled organ cultures resulted in lung injury as assessed by radioisotope release. These observations are consistent with endothelial cell culture data that indicate that TNF-induced endothelium exhibits a protein synthesis-dependent increase in adhesiveness for neutrophils. These data validate rat lung organ cultures as a model system that can be used to assess mechanisms of neutrophil adhesion and leukocyte-mediated tissue injury.

Effect of ammonium ion on pyrimidine synthesis de novo in isolated rat hepatocytes.

Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides orotic acid and the acid-hydrolyzed product of carbamoyl-aspartic acid by ion-exchange chromatography and high-pressure liquid chromatography show a marked stimulation in the incorporation of [14C]bicarbonate in incubations with added ammonium ions. The incorporation into total uridine nucleotides (sigma UMP) was increased twofold in the presence of 5 mM ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling of carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyrimidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines there was no increase in the hepatocyte content of sigma UMP, which was 11.5 mumol/g dry weight, although the orotic acid content increased from 0.09 mumol/g dry weight in the absence of added ammonium ions (but in the presence of 2 mM L-glutamine) to 8.6 mumol/g dry weight with 5 mM ammonium ion. The stimulated incorporation of [14C]bicarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80% of label in the uracil ring was present at position C-2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5% foetal calf serum and 20 mM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium ion. The stimulatory effect of ammonium ions on incorporation of bicarbonate into pyrimidines was almost completely reversed by 5 mM L-ornithine and was partially reversed by 1 mM L-ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed.

Complement component C5 modulates the systemic tumor necrosis factor response in murine endotoxic shock.

Patients with disseminated Neisseria meningitidis infections (meningococcemia) suffer from a fulminant shock syndrome that is accompanied by extraordinarily high concentrations in serum of tumor necrosis factor (TNF). People with homozygous deficiencies of late complement components (C5, C6, C7, and C8) experience a high incidence of disseminated neisserial infections yet suffer from an attenuated form of the disease. The mechanisms that account for this disparity in host response are unclear, but they may in part be related to differences in the systemic TNF response that are modulated by terminal complement components (C5 to C9). The role of C5 in the modulation of the systemic endotoxin-induced TNF response was studied with matched strains of C5-deficient (B10 D2/Osn) and complement-sufficient (B10 D2/Nsn) mice. Following lipopolysaccharide (LPS) administration, complement-sufficient mice exhibited more rapid increases in pulmonary and hepatic vascular permeabilities than did C5-deficient controls. Complement-sufficient mice developed acute passive hepatic congestion, they appeared more ill than C5-deficient mice, and they exhibited a twofold greater rise in serum TNF activity compared with that by C5-deficient mice. C5-deficient mice reconstituted with normal serum before an LPS injection exhibited pulmonary and hepatic vascular permeability increases and serum TNF levels approaching those observed in complement-sufficient mice. Alveolar and peritoneal macrophages isolated from complement-sufficient and C5-deficient mice and incubated in heat-inactivated serum did not exhibit differences in TNF mRNA expression or secreted TNF activity following stimulation with LPS. However, incubation of macrophages in complement-sufficient mouse serum (before LPS stimulation) resulted in increased TNF mRNA expression and TNF activity compared with those in cells incubated in C5-deficient serum. In vitro studies employing human complement components and peripheral blood monocytes revealed that recombinant C5a, in the presence or absence of LPS, can induce increased concentrations of TNF and that C5b to C9 had no additional modulatory effect on the TNF response. These data suggest that C5 modulates the endotoxin-triggered TNF response. The role of complement components distal to C5 (i.e., C5b to C9) in the endotoxin-triggered TNF response remains unclear.

Contrasting roles for tumor necrosis factor in the pathogeneses of IgA and IgG immune complex lung injury.

Recent studies suggest that development of acute gamma G immunoglobulin (IgG) immune complex lung injury is partially dependent on a tumor necrosis factor (TNF)-dependent mechanisms of neutrophil (PMN) recruitment. The authors have sought to further define the role of intrapulmonary TNF in IgG alveolitis and to examine its role in IgA immune complex alveolitis, a neutrophil-independent model of acute lung injury. IgG immune complex lung injury resulted in a marked rise in intrapulmonary TNF activity accompanied by progressive pulmonary PMN accumulation. Intratracheal instillation of neutralizing concentrations of anti-TNF markedly reduced PMN influx measured at 4 hours but had no effect on PMN recruitment quantitated at 2 hours. IgA immune complex deposition resulted in acute lung injury accompanied by increased numbers of intrapulmonary mononuclear phagocytes but few neutrophils. Lung lavage fluids obtained from IgA immune complex-injured rats contained both neutrophil and monocyte chemotactic activities, albeit at twofold to fourfold lower concentrations than observed in IgG-mediated alveolitis. In contrast to IgG complex-mediated alveolitis, lung lavage fluids from IgA-injured rats contained no TNF activity. Intratracheal administration of anti-TNF antibodies had no effect on the development of IgA lung injury as assessed by morphology and measurements of vascular permeability. In vitro exposure of isolated alveolar macrophages to performed IgG immune complexes resulted in dose-dependent TNF secretion, while exposure to IgA complexes resulted in very low levels of TNF secretion. These data suggest that TNF-mediated pulmonary neutrophil recruitment (in IgG lung injury) is manifest chiefly in the late phase (approximately 4 hours) of developing alveolitis. The virtual absence of intrapulmonary TNF activity in evolving IgA immune complex alveolitis may in part account for the limited PMN recruitment observed in this model.

Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.

To achieve expression of human mature apolipoprotein A-I (apoA-I) in the baculovirus-insect cell expression system, the propeptide encoding region of full-length preproapoA-I was deleted using polymerase chain reaction and the resulting cDNA was cloned into BacPak8 plasmid. After transfection into Sf21 insect cells and plaque purification, mature human apoA-I was secreted by the infected cells into the medium as determined by immunoblotting, amino-terminal sequencing, and molecular weight determination. In both monolayer cell cultures, and in suspension cell culture, maximum expression was achieved by the fifth day. For the first 4 days, 50 to 70% of the synthesized apoA-I was retained in the cells. This intracellular apoA-I was represented by mature apoA-I as shown by immunoblotting and amino-terminal sequencing. Further incubation resulted in a sharp decrease in the cell apoA-I content without a corresponding increase in protein in the medium and most likely represents intracellular degradation of the protein. We conclude that the deletion of the propeptide, while not preventing the correct cleavage of prepeptide during intracellular processing, results in reduced secretion of mature apoA-I. The baculovirus-insect cell expression system described in this study provides a useful method for producing recombinant mature apoA-I and is a potential tool for understanding the function of propeptide in intracellular transport and secretion of apoA-I from cells.

Role of intercellular adhesion molecule-1 in glucan-induced pulmonary granulomatosis in the rat.

Glucan-induced pulmonary granulomatous vasculitis in the rat mimics several human lung diseases (e.g., Wegener's granulomatosis, intravenous talcosis). We sought to clarify the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of glucan-induced granulomatous vasculitis. Immunohistochemical analysis of lung sections from rats with florid vasculitis (48 hours) revealed marked alveolar septal and lesional expression of ICAM-1. An ex vivo binding analysis with isotope-labeled antibodies and lung sections taken at various times up to 48 hours after glucan infusion revealed a progressive increase in whole-lung ICAM-1 expression. In vivo measurements of vascular wall-associated ICAM-1 expression revealed an earlier rise that began less than 6 hours after glucan infusion, peaked at 24 to 48 hours, and then declined to near baseline during the ensuing 24 to 96 hours. To assess whether ICAM-1 expression both within blood vessel walls and within lesions per se is important in granuloma development, we carried out in vivo neutralization experiments with several different routes of administration of antibody to ICAM-1. Monoclonal antibody to rat ICAM-1 was either infused intravenously at time 0 (when glucan was infused), infused intravenously at time 0 and after 24 hours, instilled only intratracheally 24 hours after glucan infusion, or given both intravenously (time = 0 and 24 hours) and intratracheally (time = 24 hours). Infusions of monoclonal antibody to rat ICAM-1 resulted in dose-dependent reductions in mean granuloma number and cross-sectional area. Intrapulmonary instillation of antibody to rat ICAM-1 (via tracheostomy 24 hours after glucan infusion) resulted in a modest reduction in mean granuloma number and cross-sectional area. When antibody to ICAM-1 was both infused and instilled via the trachea, we found an additive reduction in mean granuloma size and number. There was a 12-fold increase in adhesion of ED-1-positive peripheral blood mononuclear cells (monocytes) to granuloma-bearing frozen lung sections prepared 48 hours after glucan infusion. Moreover, 73% of the additional adherent monocytes were bound specifically to granulomas per se. The increase in ex vivo monocyte binding to lung sections prepared at 48 hours was reduced 62% when sections were incubated with monoclonal antibody to ICAM-1. Taken together, these data indicate that ICAM-1 expression in evolving glucan-induced granulomatous vasculitis occurs first within blood vessel walls and then within lesional cells per se. The in vivo blocking studies suggest that ICAM-1 expression in both anatomic sites is important in granuloma development.

Intrapulmonary tumor necrosis factor triggers local platelet-activating factor production in rat immune complex alveolitis.

Recent studies suggest that intrapulmonary tumor necrosis factor (TNF) participates in the pathogenesis of acute IgG immune complex alveolitis through a mechanism involving neutrophil polymorphonuclear leukocyte (PMN) recruitment. There are few in vivo studies that address mechanisms of TNF-dependent PMN recruitment and tissue injury. We have examined the relationship between intrapulmonary TNF and locally generated platelet-activating factor (PAF) in the development of acute alveolitis. Intratracheal instillation of IgG anti-bovine serum albumin followed by intravenous bovine serum albumin results in acute neutrophil-mediated alveolitis. Induction of IgG immune complex lung injury resulted in a marked increase in the whole lung and bronchoalveolar lavage PAF levels. Intratracheal instillation of the PAF antagonists, L-652,731 (Merck, Sharpe, and Dohme, Rahway, New Jersey) or WEB-2086 (Boehringer), attenuated pulmonary vascular leakage and PMN recruitment into the alveolar compartment. Neutralization of intrapulmonary TNF with anti-TNF antibodies reduced pulmonary vascular permeability, PMN recruitment, and whole lung PAF levels. Incubation of isolated mouse alveolar macrophages with recombinant murine TNF resulted in rapid (30 to 60 minutes), cell concentration-dependent PAF release. The presence of high concentrations of PAF in whole lungs obtained from PMN-depleted rats after immune complex deposition suggest that recruited PMN are not the predominant source of intrapulmonary PAF in this model. These data suggest that acute IgG immune complex alveolitis is in part mediated by TNF-triggered PAF production and that locally produced PAF promotes recruitment of PMN into the alveolar compartment.

Up-regulation of lung vascular ICAM-1 in rats is complement dependent.

Intrapulmonary deposition of IgG immune complexes in rats causes acute inflammatory lung injury that is neutrophil, complement, cytokines (IL-1, TNF-alpha), and intercellular adhesion molecule (ICAM-1) dependent. In the current studies involving the same model of lung injury, complement depletion or complement blockade resulted in substantial reductions in up-regulation of pulmonary vascular ICAM-1, accompanied by reduced lung injury and neutrophil accumulation. Complement depletion neither reduced the amount of immune complex deposited in lung nor the TNF-alpha content in bronchoalveolar lavage fluids. In the same model of inflammatory lung injury, neutrophil depletion (which is highly protective) did not affect up-regulation of lung vascular ICAM-1. Up-regulation of lung vascular ICAM-1 by intratracheally administered TNF-alpha was also prevented by complement depletion. These studies indicate an unexpected in vivo relationship between complement and up-regulation of lung vascular ICAM-1.

Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata.

A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.

Crystal structure of the human leukocyte Fc receptor, Fc gammaRIIa.

Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.