Histochemistry and Protein Profile of The Venom Glands of Workers of Neoponera villosa Ants (Hymenoptera: Ponerinae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

BPIFB1 (LPLUNC1) is upregulated in cystic fibrosis lung disease

Although the biology the PLUNC (recently renamed BPI fold, BPIF) family of secreted proteins is poorly understood, multiple array based studies have suggested that some are differentially expressed in lung diseases. We have examined the expression of BPIFB1 (LPLUNC1), the prototypic two-domain containing family member, in lungs from CF patients and in mouse models of CF lung disease. BPIFB1 was localized in CF lung samples along with BPIFA1, MUC5AC, CD68 and NE and directly compared to histologically normal lung tissues and that of bacterial pneumonia. We generated novel antibodies to mouse BPIF proteins to conduct similar studies on ENaC transgenic (ENaC-Tg) mice, a model for CF-like lung disease. Small airways in CF demonstrated marked epithelial staining of BPIFB1 in goblet cells but staining was absent from alveolar regions. BPIFA1 and BPIFB1 were not co-localised in the diseased lungs. In ENaC-Tg mice there was strong staining of both proteins in the airways and luminal contents. This was most marked for BPIFB1 and was noted within 2 weeks of birth. The two proteins were present in distinct cells within epithelium. BPIFB1 was readily detected in BAL from ENaC-Tg mice but was absent from wild-type mice. Alterations in the expression of BPIF proteins is associated with CF lung disease in humans and mice. It is unclear if this elevation of protein production, which results from phenotypic alteration of the cells within the diseased epithelium, plays a role in the pathogenesis of the disease.

Unusual phenotypic alteration of β amyloid precursor protein (βAPP) maturation by a new Val-715 → Met βAPP-770 mutation responsible for probable early-onset Alzheimer’s disease

We have identified a novel β amyloid precursor protein (βAPP) mutation (V715M-βAPP770) that cosegregates with early-onset Alzheimer’s disease (AD) in a pedigree. Unlike other familial AD-linked βAPP mutations reported to date, overexpression of V715M-βAPP in human HEK293 cells and murine neurons reduces total Aβ production and increases the recovery of the physiologically secreted product, APPα. V715M-βAPP significantly reduces Aβ40 secretion without affecting Aβ42 production in HEK293 cells. However, a marked increase in N-terminally truncated Aβ ending at position 42 (x-42Aβ) is observed, whereas its counterpart x-40Aβ is not affected. These results suggest that, in some cases, familial AD may be associated with a reduction in the overall production of Aβ but may be caused by increased production of truncated forms of Aβ ending at the 42 position.

Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root1

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor

Galina V. Mukamolova, Obolbek A. Turapov, Konstantin Kazarian, Miroslav Telkov, Arseny S. Kaprelyants, Douglas B. Kell and Michael Young (2002). The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Molecular Microbiology, 46 (3), 611-621. Sponsorship: BBSRC / Russian Foundation for Basic Research (grant 00-04-48691)/ WHO Global Programme for Vaccines and Immunization / Wellcome Trust RAE2008

Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 mu g mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from > 170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Immobilization of a recombinant endo-1,5-arabinanase secreted by Aspergillus nidulans strain A773

An endo-1,5-arabinanase (abnA) encoding gene from Aspergillus niveus was identified, cloned and successfully expressed in Aspergillus nidulans strain A773. Based on amino acid sequence comparison, the 34-kDa enzyme could be assigned to CAZy GH family 43. Characterization of purified recombinant endo-1,5-arabinanase (AbnA) revealed that it is active at a wide pH range (pH 4.0-7.0) and an optimum temperature at 70 degrees C. The immobilization of the AbnA was performed via covalent binding onto agarose-modified supports: glyoxyl iminodiacetic acid-Ni2+, glyoxyl amine, glyoxyl (4% and 10%) and cyanogen bromide activated sepharose. The yield of immobilization was similar on glyoxyl amine and glyoxyl (96%), and higher than glyoxyl iminodiacetic acid-Ni2+ (43%) support. The thermal inactivation of these immobilized preparations showed that the stability of the AbnA immobilized on glyoxyl 4 and 10% was improved by 4.0 and 10.3-fold factor at 70 degrees C. The half-life of glyoxyl 4% derivative at 60 degrees C was >48 h (pH 5), 9 h (pH 7) and 88 min (pH 9). The major hydrolysis product of debranched arabinan or arabinopentaose by glyoxyl agarose-immobilized AbnA was arabinobiose. (C) 2012 Elsevier B.V. All rights reserved.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Delivery of the p67 sporozoite antigen of Theileria parva by using recombinant Salmonella dublin: secretion of the product enhances specific antibody responses in cattle

The p67 sporozoite antigen of Theileria parva has been fused to the C-terminal secretion signal of Escherichia coli hemolysin and expressed in secreted form by attenuated Salmonella dublin aroA strain SL5631. The recombinant p67 antigen was detected in the supernatant of transformed bacterial cultures. Immunization trials in cattle revealed that SL5631 secreting the antigen provoked a 10-fold-higher antibody response to p67 than recombinant SL5631 expressing but not secreting p67. Immunized calves were challenged with a 80% lethal dose of T. parva sporozoites and monitored for the development of infection. Two of three calves immunized intramuscularly with the p67-secreting SL5631 strain were found to be protected, whereas only one of three animals immunized with the nonsecreting p67-expressing SL5631 strain was protected. This is the first demonstration that complete eukaryotic antigens fused to the C-terminal portion of E. coli hemolysin can be exported from attenuated Salmonella strains and that such exported antigens can protect cattle against subsequent parasite challenge.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.

A physiological product-release model for baculovirus infected insect cells

Existing models describe the product release from baculovirus infected insect cells as an unspecific protein leakage occurring in parallel with protein production. The model presented here shows that the observed product release of normally non-secreted proteins can be described through cell death alone. This model avoids the implicit non-physiological assumption of previous models that cells permeable to recombinant protein as well as trypan blue continue to produce protein. (c) 2005 Wiley Periodicals, Inc.

The Behavior of Hydroxyl Units of Synthetic Goethite and its Dehydroxylated Product Hematite

The behavior of the hydroxyl units of synthetic goethite and its dehydroxylated product hematite was characterized using a combination of Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) during the thermal transformation over a temperature range of 180-270 degrees C. Hematite was detected at temperatures above 200 degrees C by XRD while goethite was not observed above 230 degrees C. Five intense OH vibrations at 3212-3194, 1687-1674, 1643-1640, 888-884 and 800-798 cm(-1), and a H2O vibration at 3450-3445 cm(-1) were observed for goethite. The intensity of hydroxyl stretching and bending vibrations decreased with the extent of dehydroxylation of goethite. Infrared absorption bands clearly show the phase transformation between goethite and hematite: in particular. the migration of excess hydroxyl units from goethite to hematite. Two bands at 536-533 and 454-452 cm(-1) are the low wavenumber vibrations of Fe-O in the hematite structure. Band component analysis data of FTIR spectra support the fact that the hydroxyl units mainly affect the a plane in goethite and the equivalent c plane in hematite.