CHEMICAL MODIFICATION OF L-ASPARAGINASE FROM Cladosporium sp. FOR IMPROVED ACTIVITY AND THERMAL STABILITY

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (k_d), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.     

Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.     

Wood Anatomy and the Development of Interxylary Phloem of Ipomoea hederifolia Linn. (Convolvulaceae)

In Ipomoea hederifolia Linn., stems increase in thickness by forming successive rings of cambia. With the increase in stem diameter, the first ring of cambium also gives rise to thin-walled parenchymatous islands along with thick-walled xylem derivatives to its inner side. The size of these islands increases (both radially and tangentially) gradually with the increase in stem diameter. In pencil-thick stems, that is, before the differentiation of a second ring of cambium, some of the parenchyma cells within these islands differentiate into interxylary phloem. Although all successive cambia forms secondary phloem continuously, simultaneous development of interxylary phloem was observed in the innermost successive ring of xylem. In the mature stems, thick-walled parenchyma cells formed at the beginning of secondary growth underwent dedifferentiation and led to the formation of phloem derivatives. Structurally, sieve tube elements showed both simple sieve plates on transverse to slightly oblique end walls and compound sieve plates on the oblique end walls with poorly developed lateral sieve areas. Isolated or groups of two to three sieve elements were noticed in the rays of secondary phloem. They possessed simple sieve plates with distinct companion cells at their corners. The length of these elements was more or less similar to that of ray parenchyma cells but their diameter was slightly less. Similarly, in the secondary xylem, perforated ray cells were noticed in the innermost xylem ring. They were larger than the adjacent ray cells and possessed oval to circular simple perforation plates. The structures of interxylary phloem, perforated ray cells, and ray sieve elements are described in detail.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.     

Protective and survival efficacies of Rv0160c protein in murine model of Mycobacterium tuberculosis

The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) multi-gene families code for approximately 10 % of the Mycobacterium tuberculosis (Mtb) genome. These proteins are thought to be virulence factors that participate in impounding the host immune responses. While some members have been studied, the functions of most PE/PPE proteins are yet to be explored. The studies presented here have specifically characterized the roles of one of the PE proteins of Mtb, Rv0160c (PE4), in mycobacterial persistence and in prophylactic efficacy. We have expressed Rv0160c in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that the protein improves the survival of mycobacteria in macrophages and in mice. The protein has also shown its effect under physiological stress of bacteria, as evidenced by elevated expression in acidic and in hypoxic conditions. In mice, the level of Rv0160c was noticeably high during the chronic stage of tuberculosis. The seroreactivity of the protein against different categories of tuberculosis patients revealed a strong B-cell humoral response in freshly infected pulmonary tuberculosis patients. In mice, it exhibited increased IL-2, TNF, and IL-6 production. The antigenic properties of the protein directed towards the protective efficacy against the Mtb challenge. All together, our findings have identified Rv0160c as an in vivo expressed immunodominant antigen which plays a crucial role in the pathogenesis of mycobacterial disease and could prove to be a good preventive antigen for tuberculosis.     

Synthesis,characterization and in vitro biological evaluation of some novel 1,3,5-triazine–Schiff base conjugates as potential antimycobacterial agents

A series of some novel 1,3,5-triazine–Schiff base conjugates (1–32) have been synthesized and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using Alamar Blue assay and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compounds 4 (4-Methoxy-6-methyl-N-(3,4,5-trimethoxybenzylidene)-1,3,5-triazin-2-amine), 11 (4-Methoxy-6-methyl-N-(2-hydroxy-3-bromo-5-chloro-benzylidene)-1,3,5-triazin-2-amine) and 24 (4-Methoxy-6-methyl-N-(1-(2,5-dihydroxyphenyl)ethylidene)-1,3,5-triazin-2-amine) exhibited a significant activity at 3.125, 6.25 and 6.25 μg/mL, respectively, when compared with the antitubercular drugs such as ethambutol (3.125 μg/mL), pyrazinamide (6.25 μg/mL) and streptomycin (6.25 μg/mL) and it could be a potential starting point to develop new lead compounds in the fight against Mycobacterium tuberculosis H37Rv.     

Influence of substrate curvature on osteoblast orientation and extracellular matrix deposition

Background

The effects of microchannel diameter in hydroxyapatite (HAp) substrates on osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 μm diameter were created on hydroxyapatite disks. The changes in osteoblast precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter.

Results

Curvature did not impact cellular differentiation, however organized cellular orientation was achieved within the 100 and 250 μm microchannels (mc) after 6 days compared to the 12 days it took for the 500mc group, while the flat substrate remained disorganized. Moreover, the 100, 250 and 500mc groups expressed a specific shift in orientation of 17.45°, 9.05°, and 22.86° respectively in 24 days. The secreted/mineralized ECM showed the 100 and 250mc groups to have higher modulus (E) and hardness (h) (E?=?42.6GPa; h?=?1.6GPa) than human bone (E?=?13.4-25.7GPa; h?=?0.47-0.74GPa), which was significantly greater than the 500mc and control groups (p?<?0.05). It was determined that substrate curvature affects the cell orientation, the time required for initial response, and the shift in orientation with time.

Conclusions

These findings demonstrate the ability of osteoblasts to organize and mineralize differentially in microchannels similar to those found in the osteons of compact bone. These investigations could lead to the development of osteon-like scaffolds to support the regeneration of organized bone.