Genetic, structural and pharmacological characterization of polymannuronate synthesized by algG mutant indigenous soil bacterium Pseudomonas aeruginosa CMG1421.

AIMS: It was aimed to study the genetic, structural and pharmacological characteristics of polymannuronate synthesized by Pseudomonas aeruginosa CMG1421. METHODS AND RESULTS: Synthesis was analysed by transmission electron microscopy, FT/IR, 1 H-NMR and gel permeation chromatography followed by in vitro bioassays. Colony PCR followed by sequence analysis was employed for screening of structural genes. FT/IR analysis indicated the presence of hydroxyl, carboxyl and O-acetyl groups linked to mannuronate. 1 H-NMR analysis indicated M-M bond characteristics for mannuronic acid residues. The average relative molecular weight was found in range of 20 000-250 000 Da. The amplified DNA fragments were identified as 16S rRNA, algD, alg8, alg44, algG, algE and algX genes showing 99-100% homology with those of P. aeruginosa. However, in algG there were transition mutations of adenine and cytosine at nucleotide position 766 and 769, and 878 and 881 respectively. Polymannuronate and its oligomannuronates respectively showed moderate and significant antioxidant, anti-inflammatory, anti-obesity and antidiabetic activities. CONCLUSIONS: Alginate synthesized by ∆algG mutant P. aeruginosa CMG1421 was bioactive and solely consists of acetylated d-mannuronates. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated biocompatible, nonimmunogenic and nontoxic pharmacological agents for treatment and attenuation of degenerative, inflammatory, autoimmune disease, and metabolic disorders such as obesity and diabetes.

Contrast efficacy of novel phase convertible nanodroplets for safe CEUS imaging.

Microbubble contrast agents in ultrasound/echocardiography are used to increase the echogenicity of the target tissues, thereby raising the contrast resolution of the resultant image. Recently, the trend has shifted toward the development of phase-convertible nanodroplets as ultrasound contrast agents due to their promising theragnostic potential by switching capability at the active site. Herein, we fabricated pre-PGS- perfluoropentane phase convertible nanodroplets and checked their in vitro and in vivo enhancement and safety profile. For this, we performed experiments on 20 male Wistar rats and 2 dogs. Biochemical assays of both rats and dogs included complete blood profiles, liver function tests, and renal function tests. For rat vitals, monitoring and histopathological analysis were also performed. Converted nanodroplets showed excellent contrast enhancement, better than Sonovue upon in vitro testing, with an enhancement time of up to 14 min. In vivo, experiments showed comparable opacification of the ventricles of both rats and dogs. All biochemical assays remained within the normal range during the study period. The histopathological analysis did not show any signs of drug-induced toxicity, showing the safety of these nanodroplets. Pre-PGS-PFP nanodroplets hold great potential for use in echocardiography and abdominal imaging in both human and veterinary applications after clinical trials.

Production, characterization, in vitro and ex vivo studies of babchi oil-encapsulated nanostructured solid lipid carriers produced by a hot aqueous titration method.

An aqueous dispersion of solid fat nanoparticles of babchi oil (BOSLN) was prepared by means of the hot water titration method. Surface morphology was determined by HR-TEM which revealed a fairly spherical shape of the formulations. Further they were evaluated for in vitro drug release characteristics and ex vivo skin permeation profile, zeta potential and particle diameter, rheological measures and droplet size distribution. Highest values for steady state flux (Jss), permeability coefficient (Kp) and enhancement ratio (Er) were observed for formulation, BOSLN3 comprised of oil [10% v/v; BO (3.33%), CAT (6.67%)], Tween 80 (9.25% v/v), transcutol-P (28.75% v/v) and distilled water (53% v/v). These results suggest that the studied SLN might be promising vehicles for babchi oil in the management of psoriasis.

Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart.

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development.

Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies.

Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.

Muscular dystrophy: inhibition of degeneration in vivo with protease inhibitors.

The protease inhibitors leupeptin and pepstatin were used in vivo in genetically dystrophic chickens to determine their effects on the histological and biochemical changes observed in this disease. These compounds appear to delay the degeneration of muscle tissue which is characteristic of this disorder and thus may have potential therapeutic value in the treatment of muscular dystrophy.

Effect of Labrasol on self-nanoemulsification efficiency of ramipril nanoemulsion.

The purpose of the present investigation was to evaluate the capacity of Labrasol as surfactant for self-nanoemulsification efficiency of ramipril nanoemulsion formulation. Based on the solubility profile of ramipril, Sefsol-218, Labrasol and Carbitol were selected as oil phase, surfactant and cosurfactant, respectively. Based on the stability profile of ramipril, standard buffer solution of pH 5.0 was selected as an aqueous phase for the development of ramipril nanoemulsion formulation. Nanoemulsion formulations of ramipril were developed using an aqueous phase titration method. Pseudoternary phase diagrams were constructed to identify the nanoemulsion region. Selected formulations were subjected to different thermodynamic stability tests using centrifugation, heating cooling cycles and freeze thaw cycles. The formulations which were stable at thermodynamic stability tests were taken for self-nanoemulsification efficiency test. No creaming, cracking, coalescence or phase inversion was observed on most of the formulations upon thermodynamic stability tests. All the formulations passed self-nanoemulsification tests in grade C, D and E but not in grade A and B. Because none of the formulation passed self-nanoemulsification efficiency test in grade A and B, it was concluded that Labrasol is not suitable as surfactant for oral or self nanoemulsifying drug delivery system of ramipril.

Enhanced anti-inflammatory effects of celecoxib from a transdermally applied nanoemulsion.

The aim of the present study was to evaluate the enhanced anti-inflammatory effects of celecoxib (CXB) from a transdermally applied nanoemulsion. The anti-inflammatory effects of an optimized nanoemulsion formulation were compared with those of conventional CXB gel and nanoemulsion gel on carrageenan-induced paw edema in rats. These tests were compared using the Dunnett test of one-way analysis of variance (ANOVA). The % inhibition value after 24 h application was significant for optimized formulation C2 (85.4%) compared with CXB gel and nanoemulsion gel (p < 0.05). These results suggest that nanoemulsions can be successfully used to enhance the anti-inflammatory effects of CXB.

Skin permeation mechanism of aceclofenac using novel nanoemulsion formulation.

The aim of the present study was to investigate the skin permeation mechanism of aceclofenac using a novel nanoemulsion formulation. An optimized oil-in-water nanoemulsion of aceclofenac was prepared by the spontaneous emulsification method. The optimized nanoemulsion contained 2% w/w aceclofenac, 10% w/w Labrafil, 5% w/w Triacetin, 35.33% w/w Tween 80, 17.66% w/w Transcutol P and 32% w/w distilled water. The skin permeation mechanism was evaluated by FTIR spectroscopy, DSC thermography, activation energy measurement and histopathological examination. FTIR spectra of skin treated with the nanoemulsion formulation indicated breaking of the hydrogen bond network at the head of ceramides. DSC thermograms indicated that intracellular transport could be a possible mechanism of permeation enhancement and that permeation occurred due to the extraction of SC lipids by the nanoemulsion. The significant decrease in activation energy for aceclofenac permeation across rat skin indicated that the SC lipid bilayers were significantly disrupted (p < 0.05). Photomicrography of skin showed disruption and extraction of lipid bilayers as distinct voids and empty spaces visible in the epidermal region. Overall these findings indicated that nanoemulsions can be successfully used to enhance skin permeation of drugs.

Combination of needle aspiration and core needle biopsy: A new technique of stereotactic biopsy.

AIM: The study aims at describing the results of using a new technique to acquire the tissue sample in stereotactic biopsy of brain lesions. MATERIALS AND METHODS: The study was performed in 19 patients over a period of 5 years in which we used the new technique, i.e., Abrar and Afzal technique (AT) of obtaining tissue biopsy. It is a combination of core tissue biopsy and needle aspiration techniques. The technique was devised to acquire greater amount of tissue for pathologic study. RESULTS: While we could give pathologic diagnosis in 18 patients out of 19 (94.7%), in one patient, the tissue sample revealed only inflammatory cells and definitive diagnosis could not be reached. There was no significant morbidity or any mortality in the series. CONCLUSION: Abrar and Afzal technique is a reasonably accurate technique of acquiring larger tissue sample in stereotactic brain biopsy without any additional risks. It can be done with little modification of the conventional equipment available with the stereotactic system.

(CTG)n repeats markedly inhibit differentiation of the C2C12 myoblast cell line: implications for congenital myotonic dystrophy.

Although the mutation for myotonic dystrophy has been identified as a (CTG)n repeat expansion located in the 3'-untranslated region of a gene located on chromosome 19, the mechanism of disease pathogenesis is not understood. The objective of this study was to assess the effect of (CTG)n repeats on the differentiation of myoblasts in cell culture. We report here that C2C12 myoblast cell lines permanently transfected with plasmid expressing 500 bases long CTG repeat sequences, exhibited a drastic reduction in their ability to fuse and differentiate into myotubes. The percentage of cells fused into myotubes in C2 C12 cells (53.4+/-4.4%) was strikingly different from those in the two CTG repeat carrying clones (1.8+/-0.4% and 3.3+/-0. 7%). Control C2C12 cells permanently transfected with vector alone did not show such an effect. This finding may have important implications in understanding the pathogenesis of congenital myotonic dystrophy.

Beta-adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes.

BACKGROUND-Catecholamine-induced apoptosis is mediated by activation of the beta-adrenergic signaling pathway. We tested the hypothesis that beta(1)- and beta(2)-adrenergic receptor (AR) subtypes differentially affect apoptosis in adult rat ventricular myocytes in vitro. METHODS AND RESULTS-Myocytes were first exposed to norepinephrine (NE) alone (10 mcmol/L) or NE+atenolol (AT) (10 mcmol/L) for 12 hours. AT, a beta(1)-selective AR antagonist, abolished the NE-induced increase in nick end-labeling (TUNEL)-positive cells compared with control (NE, 33+/-3% versus control, 3+/-1%, P<0.0001; NE+AT, 4+/-2% versus control, 3+/-1%, P=0. 98). Annexin V staining, DNA laddering, and caspase activity determinations corroborated these results. Subsequent experiments under prazosin treatment established the apoptosis dose-response curves for the increasingly beta(2)-selective AR agonists isoproterenol (ISO) (beta(1) approximately beta(2)) and albuterol (ALB) (beta(2)>beta(1)). ISO and ALB induced significantly less apoptosis than NE (beta(1)>beta(2)) at equimolar concentrations as assessed by TUNEL staining [1 mcmol/L: NE (8+/-2%) approximately ISO (7+/-1%)>ALB (2+/-1%); 10 mcmol/L: NE (35+/-2%)>ISO (23+/-1%)>ALB (3+/-1%); 100 mcmol/L: NE (50+/-2%)>ISO (29+/-2%)>ALB (14+/-1%), P<0.0001 except for NE versus ISO at 1 mcmol/L with P=0.62]. ALB-induced apoptosis at 100 mcmol/L was abolished by AT (10 mcmol/L), indicating a beta(1)AR-mediated effect. Importantly, ICI 118551 (0.1 mcmol/L), a highly selective beta(2)AR antagonist, did not decrease the percentage of NE-, ISO-, and ALB-induced apoptosis. Reverse transcription-polymerase chain reaction studies revealed that AT completely reversed the beta-adrenergic signaling-induced changes in the Bcl-2-to-Bax ratio. CONCLUSIONS-These observations provide evidence that beta AR-mediated apoptotic death signaling is largely dissociated from beta(2)ARs and selectively mediated by beta(1)ARs in adult rat ventricular myocytes.

In situ hybridization analysis for expression of myogenic regulatory factors in regenerating muscle of mdx mouse.

Precise temporal and spatial coordination of expression of the myogenic regulatory factors (MRF) plays a critical role in the development of skeletal muscle. Whether this pattern is recapitulated postnatally during regeneration of mature muscle after injury is not known. The aim of this study was to determine the cellular distribution of mRNA expression of the various myogenic regulatory factors (MyoD, Myogenin, Myf-5 and Myf-6) during regeneration in mature skeletal muscle. We used the mdx mouse, an animal model for Duchenne muscular dystrophy, which undergoes necrosis and vigorous regeneration of muscle fibers, as a natural model to study muscle fibers at various stages of maturity ranging from satellite cells to mature muscle fibers. In situ hybridization analysis revealed that MyoD expression was the most widespread and was expressed in satellite cells and small regenerating muscle fibers surrounding necrotic fibers. Myogenin, Myf5, and Myf6 were expressed weakly in some immature fibers and none of the MRF's could be detected in mature muscle. These findings, taken together with previous studies, suggests that the pattern of expression of the various MRF's in regenerating skeletal muscle differs from that in developing muscle in embryos and that MyoD may play a critical role in the initiation and progression of events which lead to regeneration in mature skeletal muscle.

Freeze-fracture analysis of the effects of age and 2,4-dinitrophenol on the morphology of flight muscle mitochondria of Musca domestica L.

Morphometric comparison of freeze-fractured mitochondria in flight muscles of adult (37-day-old) and old (68-day-old) houseflies revealed a 28% decrease of cristae in the old flies. The major membrane change with age was an increase in the 90-120-A particles in the inner membrane external face concomitant with a loss of particle clusters associated with the openings of the cristae on to the inner membrane. In vitro treatment of flight muscle with 2,4-dinitrophenol, and uncoupler of mitochondrial respiration, did not produce this change but resulted in the formation of smooth particle-free vesicular swellings in the mitochondria. Such swelling were infrequent in the old muscle. The cause for the aging change is not clear, but a reduction in the ability of the intramembranous particles to aggregate, either through modification of altered synthesis, is indicated.

Acute myopathy with selective lysis of myosin filaments.

An acute reversible myopathy characterized by extensive lysis of myosin occurred in a patient who suffered from severe shock, hypoxia, and acidosis. This new clinical pathologic entity illustrates an unusual structural change in muscle associated with these catastrophic clinical circumstances.

Centronuclear myopathy heterogeneity: distinction of clinical types by myosin isoform patterns.

We studied muscles from 3 patients with centronuclear myopathy (CNM) by immunocytochemistry using myosin heavy chain (MHC)-specific monoclonal antibodies to determine whether subtypes of CNM express prenatal MHC and to assess if there is an arrest in development of these muscles. Muscle from a woman with childhood-onset CNM did not express prenatal MHC, yet this prenatal MHC was strongly expressed in the muscle fibers of 2 brothers with X-linked CNM. This finding represents the 1st immunocytochemical evidence of the expression of a prenatal myosin isoform in nonregenerating postnatal human muscle and suggests that the X-linked form of CNM differs from the other types because of a true arrest in maturation of the muscle.

Monoclonal antibody ALD 180: a reagent specific for a human prenatal skeletal muscle myosin isoform.

We report the characterization of monoclonal antibody (MAb) ALD 180, prepared against the myosin of slow avian muscle, for studies of human muscle development and disease. With the use of radioimmunoassays, Western immunoblots of native and denatured myosins, and epifluorescent indirect immunocytochemistry, we show that ALD 180 is specific for an epitope in human prenatal skeletal muscle myosin heavy chain (MHC), which is expressed in diminishing abundance in fetal fibers from at least 19-22 weeks' gestation to term and also in regenerating muscle fibers seen in diseased muscles from both children and adults. ALD 180 recognizes an epitope apparently different from those reacting with anti-prenatal human myosin MAb previously described, and therefore affords a complementary reagent for use in future studies of human myosin isoform expression and regulation.

A freeze-fracture study of fiber types in normal human muscle.

The sarcoplasmic reticulum (SR) and plasma membranes of Type 1 and Type 2 fibers of normal human muscle were examined by the freeze-fracture technique. Total particle counts in the SR appeared much lower than in other mammals and a packing density of about 1200 particles/micrometer 2 was found in both longitudinal and cisternal components of SR. There was no difference in particle density of Type 1 and Type 2 fibers. In freeze-fracture replicas of plasma membranes several fiber type differences were seen. The surface caveolae were uniformly distributed in Type 1 fibers whereas in Type 2 they were clustered preferentially at the I-band levels. Total density of intramembranous particles was greater in Type 1 fibers (347 +/- 68/micrometer 2 in P-face, 58 +/- 11/micrometer 2 in E face) than in Type 2 fibers (207 +/- 30/micrometer 2 in P-face, 80 +/- 9/micrometer 2 in E-face). There was a striking difference in respect to rectilinear arrays which were virtually absent in Type 1 fibers (0--2/micrometer 2) and numberous (up to 50--70/micrometer 2) in Type 2 fibers.

Characterization of a monoclonal antibody to myosin specific for mammalian and human type II muscle fibers.

We have characterized a monoclonal antibody (McAb), ALD-19, generated against slow myosin from chicken anterior latissimus dorsi (ALD) muscle for use in studies of human and animal muscle fiber types. This McAb bound selectively to the 200 kDa myosin heavy chain band in immunoblots against chicken, rat and human myosins and showed selective staining of A bands in the myofibrils. The reactivity of ALD-19 with various myosin types was quantitated by radioimmunoassays. Fiber type analysis revealed unexpected specificity of McAb ALD-19 for type II mammalian muscle fibers. This antibody should, therefore be useful for identification and quantification of normal type II fibers in human muscle biopsy specimens.

An immunocytochemical study of type I muscle fibres in developing human skeletal muscles.

An immunocytochemical study was done on the skeletal muscles of human fetuses (19-36 weeks gestation), infants and adults using a new monoclonal antibody (McAb) ALD-47. The antibody was generated against slow myosin of chicken and is specific for myosin heavy chain (MHC). In human infants and adults the type I muscle fibres are strongly reactive with this McAb and the type II fibres uniformly non-reactive. In the fetuses from 19-20 weeks gestation (in whom the fibre types are not distinguishable by the histochemical myosin ATPase test) a proportion of muscle fibres react specifically with ALD-47. Other muscle fibres at this stage react positively with a fast specific MHC McAb HM-1.2 or are negative to both ALD-47 and HM-1.2 antibodies. These McAbs, thus, identify three distinct fibre populations in the early fetal muscle which by histochemical staining appears homogeneous. The percentage of ALD-47 positive fibres increases in fetuses at later gestational periods; at all stages these fibres lack reactivity with the HM-1.2 antibody. Because of its selective fibre type reactivity in differentiating muscles, the McAb ALD-47 in conjunction with HM-1.2 should be useful in immunoaffinity fractionation and biochemical studies of myosin isoforms in developing human muscles.