Expression of Integrin ��v��6 and TGF-�� in Scarless vs Scar-forming Wound Healing

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin αvβ6 is induced during wound healing, and it can activate fibrogenic transforming growth factor β1 (TGF-β1) and anti-fibrogenic TGF-β3 that play key roles in scar formation. In this study, expression of β6 integrin and members of the TGF-β pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of β6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The αvβ6 integrin was colocalized with both TGF-β isoforms in the wound epithelium. Significantly higher expression levels of β6 integrin and TGF-β1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-β3 compared with skin. The spatio-temporal colocalization of αvβ6 integrin with TGF-β1 and TGF-β3 in the wound epithelium suggests that αvβ6 integrin may activate both isoforms during wound healing. Prolonged expression of αvβ6 integrin along with TGF-β3 in the gingival wound epithelium may be important in protection of gingiva from scar formation. (J Histochem Cytochem 57:543–557, 2009)     

Outer membrane vesicle of<Emphasis Type="Italic">Neisseria meningitidis</Emphasis> serogroup B as an adjuvant to induce specific antibody response against the lipopolysaccharide of<Emphasis Type="Italic">Brucella abortus</Emphasis> S99

Brucellosis is a worldwide zoonosis and represents one of the most important public health problems in many countries, especially around the Mediterranean basin, Middle East, India and Central and South America. Currently subunit vaccines are being considered to develop effective vaccines for human which has been evidenced by vaccines currently available against the infections such as meningococcal diseases and influenza. The application of new adjuvants of microbial origins is also underway to design subunit vaccines promoting the immune responses to the antigenic determinant(s) of the vaccine. In order to explore the efficacy ofBrucella abortus lipopolysaccharide (LPS) combined with different adjuvants and proteins (as a vaccine candidate) in the induction of immune response as an effective and long-lasting immunity againstBrucella, we evaluated the outer membrane vesicle ofNeisseria meningitidis serogroup B (GBMOMV) as a subcutaneous adjuvant and a part of a brucellosis candidate vaccine to induce high titres of specific anti-Brucella abortus S99 LPS in animal model (mice). The obtained results were compared with complete and incomplete Freund’s adjuvants (CFA and IFA). LPS+GBMOMV was the most immunogenic compound that stimulated following the first injection and increase in IgG titre of about 3.90, 3.18 and 1.58 fold higher than that produced against LPS, LPS+IFA and LPS+CFA, respectively. The highest anti-LPS IgG titre was detected two weeks after the third injection (the day 42) of LPS+GBMOMV. Purified GBMOMV can be considered as a safe subcutaneous adjuvant and a part of a candidate vaccine when combined with lipopolysaccharide ofBrucella abortus S99.     

Optimization of Non-Nutritional Factors for a Cost-Effective Enhancement of Nisin Production Using Orthogonal Array Method

In order to increase nisin production in a cost-effective manner, non-nutritional factors as well as nutritional parameters must be optimized. In this study, optimization of the most important non-nutritional factors for nisin production using orthogonal array method was performed. Optimization of temperature, agitation, age and size of inoculum, medium initial pH value and flask volume/medium volume ratio in de Man, Rogosa and Sharpe (MRS) medium in batch fermentation was accomplished. Nisin was produced by Lactococcus lactis subsp. lactis PTCC 1336 and measured by bioassay method using Micrococcus luteus PTCC 1169 as the nisin-sensitive strain. The optimum levels of non-nutritional factors for maximum nisin production and productivity were obtained as: flask volume/medium volume ratio: 5.00, medium initial pH value: 8.00, inoculum size: 1%, inoculum age: 24 h old (A = 1.7), agitation: 100 rpm and temperature: 27 °C. Under the optimized conditions, maximum nisin production and maximum nisin productivity were 599.70 IU/mL and 37.48 IU/mL/h, respectively.     

THE USE OF BEEF LIVER CATALASE AS A PROTEIN TRACER FOR ELECTRON MICROSCOPY

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.     

Peroxisomes (microbodies) in the myocardium of rodents and primates

Summary The occurrence of peroxisomes (microbodies), their cytochemical characteristics and their ultrastructural relationship to the neighboring organelles were investigated in the ventricular myocardium of four rodent (rat, rabbit, gerbil, and guinea pig) and two primate (Macaca Java and Tupaya) species. The hearts were fixed by vascular perfusion with glutaraldehyde and incubated in alkaline diaminobenzidine media for visualization of catalase. The electron-dense reaction product of catalase was found in the myocardium of all examined species and was localized in 0.2–0.5 m oval particles, surrounded by a single limiting membrane and located usually at the junction of I and A bands. The peroxisomes in the hearts of gerbil and Macaca java were especially long and tortuous. A close spatial association was found between the myocardial peroxisomes and mitochondria, lipid droplets, and the membranes of sarcoplasmic reticulum, especially the so-called junctional sarcoplasmic reticulum. These observations demonstrate the consistent occurrence of peroxisomes in the heart of various mammalian species and suggest that peroxisomes have important metabolic and physiological functions in myocardium.This study was supported by Grant 08533 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland and a grant from Sonderforschungs-bereich 90 (Cavas) of the Deutsche Forschungsgemeinschaft, Germany. Dr. Fahimi was the recipient of a Research Career Development Award from the National Institutes of Health, Bethesda, Maryland. The technical assistance of Ms. Gaby Krämer and Mr. Michel Le Hir as well as the secretarial help of Ms. Gina Folsom is gratefully acknowledged     

Soybean bacterial artificial chromosome contigs anchored with RFLPs: insights into genome duplication and gene clustering.

Surveying the soybean genome with 683 bacterial artificial chromosome (BAC) contiguous groups (contigs) anchored by restriction fragment length polymorphisms (RFLPs) enabled us to explore microsyntenic relationships among duplicated regions and also to examine the physical organization of hypomethylated (and presumably gene-rich) genomic regions. Numerous cases where nonhomologous RFLPs hybridized to common BAC clones indicated that RFLPs were physically clustered in soybean, apparently in less than 25% of the genome. By extension, we speculate that most of the genes are clustered in less than 275 M of the soybean genome. Approximately 40%-45% of this gene-rich portion is associated with the RFLP-anchored contigs described in this study. Similarities in genome organization among BAC contigs from duplicate genomic regions were also examined. Homoeologous BAC contigs often exhibited extensive microsynteny. Furthermore, paralogs recovered from duplicate contigs shared 86%-100% sequence identity.     

GLP-1 (9-36) amide, cleavage product of GLP-1 (7-36) amide, is a glucoregulatory peptide

Objective: Glucagon‐like peptide‐1 (GLP‐1) (7–36) amide is a glucoregulatory hormone with insulinotropic and insulinomimetic actions. We determined whether the insulinomimetic effects of GLP‐1 are mediated through its principal metabolite, GLP‐1 (9–36) amide (GLP‐1m). Methods and Procedures: Glucose turnover during two, 2‐h, euglycemic clamps was measured in 12 lean and 12 obese (BMI <25 or >30 kg/m²) male and female subject volunteers with normal oral glucose tolerance test. Saline or GLP‐1m were infused from 0 to 60 min in each study. Additionally, seven lean and six obese subjects underwent a third clamp in which the GLP‐1 receptor (GLP‐1R) antagonist, exendin (9–39) amide was infused from ?60 to 60 min with GLP‐1m from 0 to 60 min. Results: No glucose infusion was required in lean subjects to sustain euglycemia (glucose clamp) during saline or GLP‐1m infusions. However, in obese subjects glucose infusion was necessary during GLP‐1m infusion alone in order to compensate for a marked (>50%) inhibition of hepatic glucose production (HGP). Plasma insulin levels remained constant in lean subjects but rose significantly in obese subjects after termination of the peptide infusions. During GLP‐1R blockade, infusion of glucose was immediately required upon starting GLP‐1m infusions in all subjects due to a more dramatic reduction in HGP, as well as a delayed and modest insulinotropic response. Discussion: We conclude that GLP‐1m potently inhibits HGP and is a weak insulinotropic agent. These properties are particularly apparent and pronounced in obese but only become apparent in lean subjects during GLP‐1 (7–36) receptor blockade. These previously unrecognized antidiabetogenic actions of GLP‐1m may have therapeutic usefulness.     

GLP-1 and appetite responses to a meal in lean and overweight adolescents following exercise

Objective: Increased physical activity is an integral part of weight loss programs in adolescents. We prospectively investigated the effects of exercise on glucagon‐like peptide‐1 (GLP‐1) concentrations and on appetite markers. Methods and Procedures: Normal weight (NW) and at risk of overweight/overweight (OW) male adolescents (n = 17/gr) underwent five consecutive days of aerobic exercise (1 h/day). A test meal was administered prior to the first exercise session and 36 hours following the last exercise session. GLP‐1 and markers of appetite were assessed. Results: GLP‐1 concentrations over the course of the test meal were lower in OW compared to NW boys (P < 0.05), while fasting GLP‐1 concentrations tended to be lower in OW boys (0.05 < P < 0.1). Exercise caused an increase in the acute GLP‐1 response to the liquid meal (from 52 to 78%, P = 0.02) that was similar in NW and OW adolescents. OW adolescents expressed greater restraint compared to NW adolescents (three‐factor eating questionnaire, TFEQ) and there was a significant correlation between TFEQ for restraint score and BMI s.d. both before and after exercise intervention (P < 0.015). There was no significant correlation between markers of appetite and GLP‐1 concentrations. Discussion: Lower concentrations of GLP‐1, a satiety hormone, in OW compared to NW male adolescents support the theory that GLP‐1 plays a role in the etiology of overweight. Whether the greater meal‐induced, 0–30 min GLP‐1 response following exercise is associated with increased satiety, a potentially beneficial effect of exercise, needs to be evaluated in larger studies.     

Heterogeneous inhibition of horseradish peroxidase activity by cadmium

Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd(2+) for various periods of time. The H(2)O(2)-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd(2+) was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd(2+) concentration. Furthermore, for the same inhibition type, K(i) values dropped as incubation time increased. These results suggested that Cd(2+) would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd(2+) altered the heme Soret absorption band on binding HRP and exhibited a K(d) which decreased as the incubation time of HRP with Cd(2+) increased. Hill plots suggested a cooperative binding of up to three Cd(2+) ions per molecule of HRP. Thus, Cd(2+) binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd(2+) ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.     

Dual Function of Mitochondrial Nm23-H4 Protein in Phosphotransfer and Intermembrane Lipid Transfer: A CARDIOLIPIN-DEPENDENT SWITCH*?

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.