Nanoscaled Bionic Periosteum Orchestrating the Osteogenic Microenvironment for Sequential Bone Regeneration.

Periosteum orchestrates bone repair. Previously developed artificial periosteum was mainly focusing on materials modification to simply enhance bone formation, but few were attempting to make the artificial periosteum fit different bone repair stages. Here, we constructed a functionalized periosteum, which was composed of an electrospun scaffold grafted with leptin receptor antibody (LepR-a) and BMP2-loaded hollow MnO2 (h-MnO2) nanoparticles through a polydopamine (PDA)-assisted technique. The bionic periosteum showed suitable mechanical properties and favorable biocompatibility. It effectively recruited skeletal stem cells (SSCs) through antigen-antibody interactions, as in in vitro cell adhesion tests, we observed that more SSCs attached to the LepR-a-grafted periosteum compared to the control group. In vivo, the LepR-a-grafted periosteum covered on the cranial defect in Prx1-Cre/ERT2, -EGFP mice recruited more Prx1-EGFP cells to the fracture site compared to control groups at post-surgery day 3, 7, and 14. Co-staining with Sp7 indicated that most of the recruited Prx1-EGFP cells underwent osteogenic lineage commitment. Sustained BMP2 release from h-MnO2 promoted osteogenesis by accelerating the osteogenic differentiation of recruited SSCs, as demonstrated by alkaline phosphatase (ALP) and alizarin red staining (ARS) in vitro and microcomputed tomography (micro-CT) in vivo. Interestingly, we also observed the growth of osteogenic coupled capillaries (CD31hiEmcnhi) in the bone repair site, which might be induced by increased platelet-derived growth factor-BB (PDGF-BB) in the regenerative microenvironment subsequent to SSCs' differentiation. Taken together, the findings from this study indicate that the multifunctionalized periosteum efficiently recruited and motivated the SSCs in vivo and orchestrated the osteogenic microenvironment for bone repair in a sequence manner. Thus, the construction of the bionic periosteum to couple with natural bone regeneration stages has been demonstrated to be effective in facilitating bone healing.

Studies of the subtilin leader peptide as a translocation signal in Escherichia coli K12.

Subtilin is an antimicrobial peptide of the lantibiotic family that is produced by Gram-positive Bacillus subtilis, and its biosynthesis involves expression of presubtilin which consists of a leader segment and a mature segment. The leader segment is unlike a typical sec-type general secretion signal, and its ability to mediate translocation through a non-sec pathway has been previously studied by fusing the subtilin leader to an alkaline phosphatase reporter and expressing it in B. subtilis 168 [Izaguirre, G. and Hansen, J. N. (1997) Appl. Environ. Microbiol. 63, 3965-3971]. In this work, we have expressed the same subtilin leader-AP fusion in Gram-negative Escherichia coli, and found that the AP polypeptide is translocated into the periplasmic compartment and assembles into an enzymatically active form. The subtilin leader segment was not cleaved from this enzymatically active AP, which remained associated with the membrane. Conversion of the cells to spheroplasts followed by treatment with proteinase K showed that about 50% of the bound AP was sufficiently exposed on the surface of the spheroplasts to be inactivated by proteolytic cleavage.

Role of reactive oxygen species in renal damage in experimental leprosy.

Renal involvement is known to occur in leprosy. In the present study the possible role of reactive oxygen species (ROS) in causation of renal damage in mice infected with Mycobacterium leprae has been investigated. At least six animals from each group (control and infected) were killed at 0 day, 3, 6 and 9 months postinfection. The results showed a significant increase in the chemiluminescence (CL) response of peritoneal macrophages which was maximum between 3 and 6 months. No significant increase was observed in CL response of blood neutrophils. A significant increase in lipid peroxidation was observed at 3 and 6 months as evident by an increase in malondialdehyde levels. The increased ROS production might be the cause of lipid peroxidation. The renal damage is alos evident by decrease in the activity of renal brush border membrane enzymes, namely, alkaline phosphatase, leucine aminopeptidase and r-glutamyl transpeptidase. Thus ROS might play a role during early stages of M. leprae infection but in the later stages other immunological mechanisms may overpower the effect of ROS.

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.

Renal brushborder membrane vesicle. Study of marker enzymes and uptake of nutrients in Mycobacterium leprae infected mice.

The renal brush border membrane vesicles (BBMV) were used to elucidate the early biochemical functional status during the course of experimental M. leprae infection in mice. The activities of the characteristic brush-border enzymes viz: alkaline phosphatase, leucine amino peptidase and gamma-glutamyl transpeptidase were found to be significantly decreased (p less than 0.001) at 3 and 6 months after infection. The transport of nutrients viz: D-glucose, L-alanine, L-lysine and L-aspartate across BBMV showed similar pattern. The activity of brush border enzymes and transport of nutrients across the membrane returned to normal at 9 months post-infection suggesting regeneration of the brush border membrane.

Leucocytic alkaline phosphatase activity in leprosy: a possible guide in the follow-up of leprosy.

In order to investigate a possible involvement of phagocytic cells in the various types of leprosy, we undertook the study of enzymatic activities in circulating leucocytes. The activity of leucocytic alkaline phosphatase was studied by histochemical techniques on blood smears in 31 patients presenting with leprosy and aged between 4 and 73, and in 11 non infected people. The 31 patients suffering from leprosy were distributed as following: 14 lepromatous leprosy of which 6 had not yet been treated and 8 were under treatment, 9 cases of tuberculoid leprosy of which 7 had been treated and 2 had not yet, 3 cases of borderline leprosy which had all been treated, and 5 patients whose form of leprosy was indeterminate (before treatment). The distribution of the different values we obtain shows a very significant difference (p less than 0.001) between patients with and without leprosy (respectively 33.8 +/- 7.3 and 109.8 +/- 12.5). Moreover, the decrease of the alkaline phosphatase activity correlated with the severity of the disease (47.2 +/- 11.4 in tuberculoid leprosy and 20.6 +/- 9.3 in lepromatous leprosy) thus suggesting that the evaluation of leucocytic alkaline phosphatase activity should be advised as a possible prognosis guide in indeterminate leprosy.

Alkaline phosphatase activity in peripheral nerves and endothelial cell cultures in leprosy.

Alkaline phosphatase activity in leprosy nerves was studied. The activity was mainly in blood vessels and was maximum in healthy nerves. Low levels were seen in crush injury. In leprosy lower levels were in BT than LL cases. Endothelial cells (in vitro) released alkpase when infected with live bacilli only. No response was observed with heat killed bacilli.

Properties of alkaline phosphatase of the halotolerant yeast Debaryomyces hansenii.

The molecular weight of a partially purified alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) from the halotolerant yeast Debaryomyces hansenii was estimated to 110,000 by gel filtration. The isoelectric point determined by electrofocusing was at approximately pH 4.4. The enzyme had a broad specificity against phosphomonoesters and also attacked some acid anhydrides. Arsenate, molybdate, and orthophosphate acted as competitive inhibitors. Various metal-binding agents inhibited enzyme activity. A zinc addition almost completely reversed the EDTA inhibition. Magnesium stimulated enzyme activity and was required for maintenance of activity at high concentrations of Na+. Increasing glycerol concentration increased the value of the Michaelis constant (Km) and decreased the maximum velocity (V). Solutions equimolar in KCl and NaCl stimulated enzyme activity by increasing V, whereas the Km was almost unaffected by salt concentration. Enzyme extracted from cells cultured at low salinity was indistinguishable from that of cells grown in the presence of 2.7 M NaCl with respect to several criteria.