Studies on the intracellular degradation of newly synthesized collagen in 3-methylcholanthrene induced fibrosarcoma cells.

The intracellular degradation of newly synthesized collagen was studied in both normal fibroblast and 3-methylcholanthrene induced fibrosarcoma cells. The degradation of newly synthesized collagen was examined using pulse-chase experiments and radioactive labelling techniques with [3H]-proline. The percentage of intracellular proteolysis of newly synthesized collagen was determined by measuring the formation of [3H]-hydroxyproline containing fragments in alcohol-soluble and insoluble fractions of normal cells and fibrosarcoma cells in the culture. The rate of degradation of newly formed collagen was then followed by estimating the radioactivity of [3H]-hydroxyproline at different intervals, during the chase period. The results clearly demonstrated that the percent of intracellular degradation of newly synthesized collagen was approximately three fold higher in fibrosarcoma cells than in normal fibroblast cells. The increased intracellular degradation of newly formed collagen was followed by an increase in the activity of cathepsin B and L in fibrosarcoma cells. The pulse-chase experiments indicated that the rate of degradation of newly synthesized collagen in fibrosarcoma cells is relatively greater than in normal fibroblast cells. In addition, as the labelling time increased, the formation of [3H]-hydroxyproline containing peptides in the ethanol-soluble fraction were found to be increased in both normal cells and fibrosarcoma cells, but the extent of formation was higher in fibrosarcoma cells compared to normal fibroblast cells. The results of this investigation collectively suggest that the intracellular degradation of newly synthesized collagen is enhanced in fibrosarcoma cells.     

Clinical and veterinary isolates of Salmonella enterica serovar enteritidis defective in lipopolysaccharide O-chain polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.     

Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d

We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.     

Cytosolic delivery of macromolecules. II. Mechanistic studies with pH-sensitive morpholine lipids

Drug carriers containing weak acids or bases can promote cytosolic delivery of macromolecules by exploiting the acidic pH of the endosome. We have prepared two pH-sensitive mono-stearoyl derivatives of morpholine, one with a (2-hydroxy) propylene (ML1) linker and the other, an ethylene (ML2) linker. The pK(a) values of lipids ML1 and ML2, when incorporated into liposomes, are 6.12 and 5.91, respectively. Both lipids disrupt human erythrocytes at pH equal to or below their pK(a) but show no such activity at pH 7.4. Confocal microscopy studies suggest partial endosome-to-cytosol transfer of fluorescent dextran (MW 10 kDa) encapsulated in liposomes that contained 20 mol% of morpholine lipids. Interestingly, co-incubation of morpholine lipids in free or micellar form (without liposomal incorporation) with dextran resulted in efficient cytosolic delivery. Upon acidification to the endosomal pH, liposomes containing ML1 revealed: (a). leakage of entrapped solute that is independent of solute size; (b). lack of liposomal collapse into micelles as evidenced by photon correlation spectroscopy and UV light scattering; and (c). minimal inter-bilayer interactions as shown in a fluorescence resonance energy transfer assay. These observations are consistent with progressive intravesicular reorganization of lipids into stable liposomes of smaller size, but of more homogeneous distribution, upon acidification. The results emphasize a need to manipulate liposomal formulations containing ML1 such that ML1 will promote catastrophic collapse of liposomes to mixed micelles upon exposure to acidic pH. It is only then that micelle-mediated permeabilization of the endosomal membrane will lead to efficient cytosolic delivery of macromolecules originally loaded in liposomes.     

Cytosolic delivery of macromolecules. 3. Synthesis and characterization of acid-sensitive bis-detergents

A serious limitation that precludes utilization of single-tailed, pH-sensitive detergents for the cytosolic delivery of macromolecules is their low limit of incorporation in stable liposomal formulations. To address this issue, we have prepared two Gemini surfactants or 'bis-detergents' by cross-linking the headgroups of single-tailed, tertiary amine detergents through oxyethylene (BD1) or acid-labile acetal (BD2) moieties. The membrane-bound pK(a) of the twin tertiary amine headgroups was determined to be 6.37 +/- 0.36 using a fluorescence-based assay. As evidenced by thin-layer chromatography, BD2was hydrolyzed under acidic conditions (pH 5.0) with an approximate half-life of 3 h at 37 degrees C, while BD1 remained stable. Low pH-induced collapse of liposomes containing acid-labile BD2 into micelles was more facile than that of BD1. With BD1, the process appeared to be reversible in that aggregation of micelles was observed at basic pH. The irreversible lamellar-to-micellar transition observed with BD2-containing liposomes can possibly be attributed to acid-catalyzed hydrolysis of the acetal cross-linker, which generates two detergent monomers within the bilayer. Liposomes composed of 75 mol % bis-detergent and 25 mol % phosphatidylcholine were readily prepared and could entrap macromolecules such as polyanionic dextran of MW 40 kDa with moderate efficiency. The ability of BD2-containing liposomes to promote efficient cytosolic delivery of antisense oligonucleotides was confirmed by (a) their diffuse intracellular distribution seen in fluorescence micrographs, and (b) the up-regulation of luciferase in an antisense functional assay. The low pH-responsive, bis-detergent constructs described herein are suitable for triggered release strategies targeted to acidic intracellular or interstitial environments.     

Bare fiber Bragg grating immunosensor for real‐time detection of Escherichia coli bacteria

Escherichia coli (E. coli) bacteria have been identified to be the cause of variety of health outbreaks resulting from contamination of food and water. Timely and rapid detection of the bacteria is thus crucial to maintain desired quality of food products and water resources. A novel methodology proposed in this paper demonstrates for the first time, the feasibility of employing a bare fiber Bragg grating (bFBG) sensor for detection of E. coli bacteria. The sensor was fabricated in a photo‐sensitive optical fiber (4.2 µm/80 µm). Anti‐E. coli antibody was immobilized on the sensor surface to enable the capture of target cells/bacteria present in the sample solution. Strain induced on the sensor surface as a result of antibody immobilization and subsequent binding of E. coli bacteria resulted in unique wavelength shifts in the respective recording of the reflected Bragg wavelength, which can be exploited for the application of biosensing. Functionalization and antibody binding on to the fiber surface was cross validated by the color development resulting from the reaction of an appropriate substrate solution with the enzyme label conjugated to the anti‐E. coli antibody. Scanning electron microscope image of the fiber, further verified the E. coli cells bound to the antibody immobilized sensor surface.

     

Fiber bragg grating sensor based device for simultaneous measurement of respiratory and cardiac activities

This paper reports a novel optical ballistocardiography technique, which is non‐invasive, for the simultaneous measurement of cardiac and respiratory activities using a Fiber Bragg Grating Heart Beat Device (FBGHBD). The unique design of FBGHBD offers additional capabilities such as monitoring nascent morphology of cardiac and breathing activity, heart rate variability, heart beat rhythm, etc., which can assist in early clinical diagnosis of many conditions associated with heart and lung malfunctioning. The results obtained from the FBGHBD positioned around the pulmonic area on the chest have been evaluated against an electronic stethoscope which detects and records sound pulses originated from the cardiac activity. In order to evaluate the performance of the FBGHBD, quantitative and qualitative studies have been carried out and the results are found to be reliable and accurate, validating its potential as a standalone medical diagnostic device. The developed FBGHBD is simple in design, robust, portable, EMI proof, shock proof and non‐electric in its operation which are desired features for any clinical diagnostic tool used in hospital environment.

     

Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo

A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.     

miR-145-5p targets paxillin to attenuate angiotensin II-induced pathological cardiac hypertrophy via downregulation of Rac 1, pJNK,p-c-Jun,NFATc3, ANP and by Sirt-1 upregulation

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3′UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3′UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy