Characterization of the role of the Escherichia coli periplasmic chaperone SurA using differential proteomics

Little is known on how β‐barrel proteins are assembled in the outer membrane (OM) of Gram‐negative bacteria. SurA has been proposed to be the primary chaperone escorting the bulk mass of OM proteins across the periplasm. However, the impact of SurA deletion on the global OM proteome has not been determined, limiting therefore our understanding of the function of SurA. By using a differential proteomics approach based on 2‐D LC‐MSⁿ, we compared the relative abundance of 64 OM proteins, including 23 β‐barrel proteins, in wild‐type and surA strains. Unexpectedly, we found that the loss of SurA affects the abundance of eight β‐barrel proteins. Of all the decreased proteins, FhuA and LptD are the only two for which the decreased protein abundance cannot be attributed, at least in part, to decreased mRNA levels in the surA strain. In the case of LptD, an essential protein involved in OM biogenesis, our data support a role for SurA in the assembly of this protein and suggest that LptD is a true SurA substrate. Based on our results, we propose a revised model in which only a subset of OM proteins depends on SurA for proper folding and insertion in the OM.     

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).     

Oncomodulin/Truncated Protamine-Mediated Nogo-66 Receptor Small Interference RNA Delivery Promotes Axon Regeneration in Retinal Ganglion Cells

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.     

Experimental infection of Indonesia Taenia (Samosir strain) in domestic animals

Three 58-day old Small-Ear-Miniature (SEM) pigs, six 36-day old Landrace-Small-Ear-Miniature (L-SEM) pigs, and two 5-day old Holstein calves were each fed 3000 or 30,000 Indonesia Taenia (Samosir strain) eggs and sacrificed 27-195 days after inoculation. A total of 4922 cysticerci were recovered only from the livers of the three SEM pigs (1977 cysticerci) and six L-SEM pigs (2945 cysticerci). The infection rate in pigs was 100%. Cysticerci recovery rates of SEM and L-SEM pigs were 22.0% and 1.6%, respectively. Calves were not susceptible to Indonesia Taenia. More cysticerci were found in the liver parenchyma (L-SEM, 66.4%; SEM, 76.2%) than on the liver surface (L-SEM, 33.6%; SEM, 23.8%) of the infected animals. Most (99.86%) of the cysticerci recovered from the livers of L-SEM pigs were degenerated or calcified but 77.9% of those in the livers of SEM pigs were mature and only 22.1% were degenerated or calcified. Measurements of length, width, diameters of protoscolex, rostellum, and sucker and hooklet pattern indicated that Indonesia Taenia is very similar to Taiwan Taenia and very different from Taenia saginata and Taenia solium. The present findings indicate that Indonesia Taenia and Taiwan Taenia may be the same new species.     

Ultrastructural changes in rat livers perfused in vitro and in vivo with a high dose of methotrexate

Methotrexate is an antifolate that is widely used in the treatment of malignant tumours and other diseases. The present study was undertaken to examine the short-term effects of high doses of methotrexate (HD-MTX) on the ultrastructure and metabolic activity of isolated rat livers. The authenticity of the drug-induced changes was substantiated by the concomitant use of in vivo experiments. Isolated rat livers were infused with HD-MTX via the portal vein for 3 hours (total dose for each liver 2000 mg). For in vivo experiments, each rat received a single intravenous injection of a maximum tolerated dose of MTX (100 mg/kg body weight) that allowed the animals to survive for 3 days. At the end of each experimental period, MTX-treated and control livers were processed for light microscopy (LM), scanning (SEM) and transmission electron (TEM) microscopy. Oxygen consumption and thyroxine metabolism were measured in treated and control isolated livers. With the exception of a few minor differences, the structural changes in the hepatocytes after MTX treatment in vitro and vivo were similar. There were focal changes consisting of disruption of normal hepatic plates and swelling and vacuolation of the hepatocytes, with no clear evidence of restriction to a specific hepatic zone. SEM revealed striking changes in the plasma membrane, the microvillar system, intercellular junctions and the sinusoidal endothelium. TEM revealed disorganized endoplasmic reticulum, dispersion of the polyribosomes, a variety of mitochondrial changes, and glycogen redistribution. In MTX-treated isolated rat livers, the uptake of tetraiodothyronine (T4) was not affected, but triiodothyronine (T3) release was impaired. Oxygen consumption was increased in livers treated with MTX. Employing an organotypic liver perfusion model in conjunction with the in vivo experiment and the use of SEM, TEM and hepatic thyroxine measurements, this investigation revealed that infusion of HD-MTX induced early ultrastructual changes in cell membrane, intercellular junctions and cell organelles and disturbance in the functional integrity of the hepatocytes in isolated rat liver.     

Anti-inflammatory and analgesic activity of ononitol monohydrate isolated from Cassia tora L. in animal models

Ononitol monohydrate (OM) was isolated from Cassia tora L. leaves. The anti-inflammatory and analgesic activities of OM have been examined in male Wistar rats and mice. The efficacy of OM against inflammation was studied by using carrageenan-induced paw oedema, croton oil-induced ear oedema, acetic acid-induced vascular permeability, cotton pellet-induced granuloma and adjuvant-induced arthritis. The analgesic activity of OM was assessed using the acetic acid-induced abdominal constriction response, formalin-induced paw licking response and the hot-plate test. In acute type inflammation models, maximum inhibitions of 50.69 and 61.06% (P < .05) were noted with 20 mg/kg of OM in carrageenan-induced hind paw oedema and croton oil-induced ear oedema, respectively. Treatment of OM (20 mg/kg) meaningfully (P < .05) reduced the granuloma tissue formation by cotton pellet study at a rate of 36.25%. OM (20 mg/kg) inhibited 53.64% of paw thickness in adjuvant-induced arthritis model. OM has also been produced significant (P < .05) analgesic activity in acetic acid-induced abdominal constriction response, formalin-induced paw licking response and in hot-plate test suggesting its peripheral and central analgesic potential. The outcomes of the present study proposed that OM influenced on the anti-inflammatory and analgesic activities.     

Outer membrane proteome of Actinobacillus pleuropneumoniae: LC-MS/MS analyses validate in silico predictions

The Gram-negative bacterial pathogen Actinobacillus pleuropneumoniae causes porcine pneumonia, a highly infectious respiratory disease that contributes to major economic losses in the swine industry. Outer membrane (OM) proteins play key roles in infection and may be targets for drug and vaccine research. Exploiting the genome sequence of A. pleuropneumoniae serotype 5b, we scanned in silico for proteins predicted to be localized at the cell surface. Five genome scanning programs (Proteome Analyst, PSORT-b, BOMP, Lipo, and LipoP) were run to construct a consensus prediction list of 93 OM proteins in A. pleuropneumoniae. An inventory of predicted OM proteins was complemented by proteomic analyses utilizing gel- and solution-based methods, both coupled to LC-MS/MS. Different protocols were explored to enrich for OM proteins; the most rewarding required sucrose gradient centrifugation followed by membrane washes with sodium bromide and sodium carbonate. This protocol facilitated our identification of 47 OM proteins that represent 50% of the predicted OM proteome, most of which have not been characterized. Our study establishes the first OM proteome of A. pleuropneumoniae.     

Isolation and characterization of collagen from marine fish (Thunnus obesus)

In the present study, we isolated collagen from Thunnus obesus bone, which was physiochemically characterized. Two different kinds of methods were used to isolate the collagen; they are the Acid Soluble Collagen (ASC) and Acid Soluble Enzyme Collagen (ASEC) methods. The isolated collagen was characterized with Fourier Transform Infrared Spectroscopy (FT-IR), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Optical Microscopy (OM) and Scanning Electron Microscopy (SEM). FT-IR results revealed the presence of collagen. SEM and OM results depicted that collagen was in the form of fiber sponge-like scaffolds. The isolated collagen scaffold was checked with pre-osteoblast (MC3T3-E1) cell line for biocompatibility. The in vitro results revealed that the collagen scaffolds were highly biocompatible and nontoxic in nature. Herewith, we are suggesting that marine fish-derived collagen will be an excellent material for leather, film industry, pharmaceutical, cosmetics, biomedical and food applications.     

A novel polymeric flocculant based on polyacrylamide grafted carboxymethylstarch

Novel functionalized polymeric flocculants based on polyacrylamide grafted carboxymethylstarch (CMS-g-PAM) have been successfully synthesized via conventional method (using ceric ammonium nitrate as free radical initiator, in an inert atmosphere) as well as by using microwave irradiation (‘microwave initiated’ synthesis). Under optimal grafting conditions, 50% grafting has been observed in case of the microwave irradiation based method and 47% grafting has been observed in case of the conventional process. The synthesized graft copolymers have been characterized by elemental analysis, FTIR spectroscopy, intrinsic viscosity measurement, molecular weight determination, ¹³C NMR spectroscopy and scanning electron micrograph (SEM); taking carboxymethylstarch (CMS) as reference. The effects of reaction parameters onto the percentage of grafting have been studied. Further, the applicability of these grafted polymers as flocculants for the treatment of municipal sewage wastewater has also been investigated.     

The double life of a bacterial lipoprotein

It has been known for many years that the small lipoprotein Lpp, which is the most abundant protein in E. coli, exists in two forms. The 'bound' form of the protein is tethered to the outer membrane (OM) by its N-terminal lipid moiety and covalently attached to the cell wall by its C-terminal lysine residue. The exact location of the 'free' form, however, has never been determined. In this issue of Molecular Microbiology, Cowles et al. demonstrate that the free form of Lpp is an integral OM protein whose C-terminus is exposed on the cell surface. The new study provides the first example of a lipoprotein that has a dual localization and adds to a growing body of evidence that lipoproteins can span the OM despite the lack of an obvious transmembrane segment. Furthermore, the new results raise intriguing questions about the assembly of both lipoproteins and other types of OM proteins.     

Extrusion of pectin and glycerol with various combinations of orange albedo and starch

Microstructural and mechanical properties of extruded pectin and glycerol films with various combinations of orange albedo and starch were determined by universal mechanical testing (UMT), dynamic mechanical analysis (DMA), optical microscopy (OM) and scanning electron microscopy (SEM). A glass transition and a second order transition attributed to the onset of translational motion of the pectin molecules was observed in all films. Observation by OM suggested that extrusion in the presence of dilute HCl was more effective in disintegrating albedo than either water or dilute citric acid. UMT, DMA and SEM analysis revealed that extruded pectin/albedo/starch/glycerol films provided better mechanical properties than pectin/albedo/glycerol films and were comparable in mechanical properties to extruded pectin/starch/glycerol films.     

Type V secretion: mechanism(s) of autotransport through the bacterial outer membrane

Autotransport in Gram-negative bacteria denotes the ability of surface-localized proteins to cross the outer membrane (OM) autonomously. Autotransporters perform this task with the help of a β-barrel transmembrane domain localized in the OM. Different classes of autotransporters have been investigated in detail in recent years; classical monomeric but also trimeric autotransporters comprise many important bacterial virulence factors. So do the two-partner secretion systems, which are a special case as the transported protein resides on a different polypeptide chain than the transporter. Despite the great interest in these proteins, the exact mechanism of the transport process remains elusive. Moreover, different periplasmic and OM factors have been identified that play a role in the translocation, making the term 'autotransport' debatable. In this review, we compile the wealth of details known on the mechanism of single autotransporters from different classes and organisms, and put them into a bigger perspective. We also discuss recently discovered or rediscovered classes of autotransporters.     

Interdigital and foot fungal infection in patients with onychomycosis

In patients with onychomycosis (OM) 71.5% of them have been reported with plantar fungal infection. The aim of this study was to study the frequency and distribution of plantar and interdigital affection in diabetic patients and in a control group without diabetes, all of them with OM. Diabetic patients with OM were more frequently diagnosed with plantar (61.2%) than interdigital (46.7%) infection. In the control group similar results were obtained; patients with OM in 76.5% had plantar mycotic infection and 67.1% interdigital involvement.     

Intrauterine position affects sex-accessory biochemistry in adult female mice

Female mouse fetuses that develop in utero implanted between two male fetuses (2M females) are shown as adults to have significantly elevated (P less than 0.01) levels of beta-glucuronidase activity in their preputial glands compared to that of OM females, females that had not been contiguous to males in utero, and 1M females, females contiguous in utero to only one male (mean +/- SEM, 2M females = 23,9 +/- 2.1, 1M females = 13.3 +/- 5.2, and OM females = 7.8 +/- 2.5 Modified Sigma Units/mg frozen weight). It is hypothesized that the increased enzymatic activity in the preputial glands of 2M females could be important in releasing steroid metabolites from voided urine. These metabolites could then act as pheromones and thus explain some of the described differences in sexual behavior correlated with intrauterine position.     

Morphologic, morphometrical, and histological aspects of the eggs of four species of the genera Meccus (Hemiptera, Reduviidae, Triatominae)

The objective of this work was to analyze the morphological, morphometrical, and histological characteristics of eggs of four triatomine bugs species still not studied, in order to understand phylogenetic aspects and to facilitate parameters used in taxonomy, with the purpose of a specific and generic characterization of these vectors in public health. The eggs of each species had come from the collections of the Laboratory of Triatomine bugs and Culicid mosquitoes of the Faculdade de Saúde Pública - USP (Faculty of Public Health/USP). The morphologic studies were carried out through optic microscopy (OM) and scanning electronic microscopy (SEM). The eggs were measured with the help of the digital paquimeter Starrett 727. In relation to histological measures, Microtome Leica RM 2145 was used. Similarities were evidenced in the exocorial architecture of the eggs when they were studied by OM and SEM. This similarity among the eggs suggests a recent speciation, probably derived from common ancestry, representing a monophyletic group. Some structures were also detected by the histological cuts. The study has contributed to the magnification and recognition in generic and specific terms of the Triatominae subfamily. These new data will be able to subsidize a better understanding to determine roles for each vector species and to facilitate parameters to be used in taxonomy.     

The utility of human dedifferentiated fat cells in bone tissue engineering in vitro

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a α-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a α-TCP/CS were well spread and covered the α-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the α-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the α-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an α-TCP/CS may be an attractive option for bone tissue engineering.     

Glucagon-stimulated calcium efflux in the isolated perfused rat liver is dependent on cellular redox potential

In the absence of any exogenous substrates, glucagon (1 X 10(-9) M) stimulated 45Ca2+ efflux from perfused livers derived from fed rats but not in livers of 24-h-fasted animals. In livers of 24-h-fasted animals perfused under conditions which would decrease cellular NAD(P)H/NAD(P)+ ratio (pyruvate (2.0 mM) or acetoacetate (10.0 mM], glucagon (1 X 10(-9) M) did not stimulate 45Ca2+ efflux. Similarly, in livers of 24-h-fasted animals perfused with substrates which increase cellular NAD(P)H content (lactate (2.0 mM) or beta-hydroxybutyrate (10.0 mM], glucagon (1 X 10(-9) M) did not increase 45Ca2+ efflux. Glucagon (1 X 10(-9) M) elicited an increase in 45Ca2+ efflux from livers of 24-h-fasted animals, only when the livers were perfused with [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate] ratios similar to those reported for livers of fed rats. Stimulation of 45Ca2+ efflux elicited by either 8-CPT-cAMP, a cAMP analog, or high glucagon concentrations (1 X 10(-8) M) was not affected whether livers were perfused with pyruvate (2.0 mM) or lactate (2.0 mM). Administration of isobutylmethylxanthine (50 microM) alone, or glucagon (1 X 10(-9) M) in the presence of isobutylmethylxanthine (50 microM) stimulated 45Ca2+ efflux from livers of 24-h-fasted animals perfused with pyruvate (2.0 mM) but not from livers perfused with lactate (2.0 mM). The ability of glucagon (1 X 10(-9) M) to elevate tissue cAMP levels was also regulated by the oxidation-reduction state of the livers. The data indicate that glucagon-stimulated 45Ca2+ efflux from perfused livers is mediated via cAMP and is dependent on the oxidation-reduction state of the livers.     

Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains

“Autodisplay technology” is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli.