Role of Rho kinases in abnormal and normal hematopoiesis

PURPOSE OF REVIEW: Rho kinases (ROCKs) are involved in regulating a variety of physiologic functions including cytoskeletal reorganization, migration, adhesion, survival and proliferation. They do so via activating several different downstream substrates such as myosin light chain phosphatase, LIM kinase and ezrin/radixin/moesin proteins. To date, most of the conclusions with regard to the function of ROCKs have involved the use of cell line models, pharmacologic inhibitors and dominant negative approaches. Importantly, the role of ROCK in hematopoiesis or leukemogenesis in the context of whole organism remains poorly understood. RECENT FINDINGS: Recent studies utilizing mice deficient in the expression of ROCK1 have begun to shed some light into the physiologic role(s) of ROCK in both normal and abnormal hematopoiesis. Findings, thus far, suggest that ROCK plays an essential role in regulating growth and survival in different hematopoietic lineages via distinct mechanisms, in part, by utilizing distinct downstream substrates including maintaining the activation of tumor-suppressor genes. SUMMARY: In blood cells, emerging data suggest that ROCK plays an essential role in negatively regulating inflammatory and erythropoietic stress and positively regulates the growth and survival of leukemic cells

Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe \u3ci\u3eThermotoga maritima\u3c/i\u3e

Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritima. IMPORTANCE: The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima. The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production. Includes supplemental material

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of \u3ci\u3eThermotoga maritima \u3c/i\u3e

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H2 production limit. The study generated a kinetic model explaining H2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H2 production by Tma200 (0.217 mol-H2 g–1-biomass) and Tma100 (0.147 mol-H2 g–1-biomass) were higher than wild type (0.096 mol-H2 g–1 -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h–1) and Tma100 (0.495 h–1) are compared with the wild type (0.663 h–1). A higher maintenance energy along with growth and non-growth H2 coefficients corroborate the higher H2 productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H2 production. (Inludes 2 supplemental figures

Comparative kinetic modeling of growth and molecular hydrogen overproduction by engineered strains of \u3ci\u3eThermotoga maritima \u3c/i\u3e

Thermotoga maritima is an anaerobic hyperthermophilic bacterium known for its high amounts of hydrogen (H2) production. In the current study, the kinetic modeling was applied on the engineered strains of T. maritima that surpassed the natural H2 production limit. The study generated a kinetic model explaining H2 overproduction and predicted a continuous fermentation system. A Leudking-Piret equation-based model predicted that H2 production by Tma200 (0.217 mol-H2 g–1-biomass) and Tma100 (0.147 mol-H2 g–1-biomass) were higher than wild type (0.096 mol-H2 g–1 -biomass) with reduced rates of maltose utilization. Sensitivity analysis confirmed satisfactory fitting of the experimental data. The slow growth rates of Tma200 (0.550 h–1) and Tma100 (0.495 h–1) are compared with the wild type (0.663 h–1). A higher maintenance energy along with growth and non-growth H2 coefficients corroborate the higher H2 productivity of the engineered strains. The modeled data established a continuous fermentation system for the sustainable H2 production. (Inludes 2 supplemental figures

Role of intracellular tyrosines in activating KIT induced myeloproliferative disease

Gain-of-function mutations in KIT receptor in humans are associated with gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), and acute myelogenous leukemia (AML). The intracellular signals that contribute to oncogenic KIT induced myeloproliferative disease (MPD) are poorly understood. Here, we show that oncogenic KITD814V induced MPD occurs in the absence of ligand stimulation. The intracellular tyrosine residues are important for KITD814V induced MPD, albeit to varying degrees. Among the seven intracellular tyrosines examined, tyrosine 719 alone plays a unique role in regulating KITD814V induced proliferation and survival in vitro, and MPD in vivo. Importantly, the extent to which AKT, ERK and Stat5 signaling pathways are activated via the seven intracellular tyrosines in KITD814V impacts the latency of MPD and severity of the disease. Our results identify critical signaling molecules involved in regulating KITD814V induced MPD, which might be useful for developing novel therapeutic targets for hematologic malignancies involving this mutation

Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (\u3ci\u3earaA\u3c/i\u3e) in the Hyperthermophilic Bacterium \u3ci\u3eThermotoga maritima\u3c/i\u3e

Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H2). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H2 using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H2 formation and life at an extremely high temperature in the bacterial domain

ROCK1 via LIM kinase regulates growth, maturation and actin based functions in mast cells

Understanding mast cell development is essential due to their critical role in regulating immunity and autoimmune diseases. Here, we show how Rho kinases (ROCK) regulate mast cell development and can function as therapeutic targets for treating allergic diseases. Rock1 deficiency results in delayed maturation of bone marrow derived mast cells (BMMCs) in response to IL-3 stimulation and reduced growth in response to stem cell factor (SCF) stimulation. Further, integrin-mediated adhesion and migration, and IgE-mediated degranulation are all impaired in Rock1-deficient BMMCs. To understand the mechanism behind altered mast cell development in Rock1-/- BMMCs, we analyzed the activation of ROCK and its downstream targets including LIM kinase (LIMK). We observed reduced activation of ROCK, LIMK, AKT and ERK1/2 in Rock1-deficient BMMCs in response to SCF stimulation. Further, loss of either Limk1 or Limk2 also demonstrated altered BMMC maturation and growth; combined deletion of both Limk1 and Limk2 resulted in further reduction in BMMC maturation and growth. In passive cutaneous anaphylaxis model, deficiency of Rock1 or treatment with ROCK inhibitor Fasudil protected mice against IgE-mediated challenge. Our results identify ROCK/LIMK pathway as a novel therapeutic target for treating allergic diseases involving mast cells

Establishment of Standardization Parameters for Herbal Formulation, Swarasa Bhvit Amalaka Churnam

Herbal formulations are very useful for human health because of their natural and simple processing. A quality herbal formulation must pass the test for their characteristic properties. Inspite of the large number of Ayurvedic formulations are available in the market, standards of their quality are yet to be laid. Amlaka churnam or amalaki rasayanam is a swarasa bhavita, important herbal formulation used for treatment related to immunity systems of human body. In addition to this it is also used for nurturing of skin, heart, eye and stomach. The main ingredient of amalaka churnam is amla (Embalica officinalis) family Euphorbiaceae. Though amalaka churnam is described in many classical Ayurvedic granthas, but there are minor differences in their formulations. Also marketed formulations of amalaki rasayanam by manufacturers do not have description about its standardization.  Therefore, it is necessary to develop standard parameters for establishment of quality of the formulation. In this work, a standardization of herbal formulations of amalaka churnam is reported in terms of organoleptic evaluation of ingredients with rasayana formulation, loss of drying analysis, foreign matter, phytochemical screening, pH estimation, evaluation of ash value, extractive value and rheological evaluation. Keywords: Bhvit, amalaka Churnam, Organoleptic, rheological etc

KIRGANELIA RETICULATA (POIR) BAILL.-A REVIEW ON ITS BOTANY, ETHNOBOTANY, PHYTOCHEMISTRY AND PHARMACOLOGY

Ethnopharmacological relevance: Kirganelia reticulata (Poir.) Baill. is known for its importance in various traditional medicine around the world and are proved pharmacologically as an antiviral against Hepatitis B, hepatoprotective, antidiabetic and antioxidant. In Ayurvedic system of Indian medicine recognized its activity against jaundice, diuretic, fever, liver disorder, in bleeding gums, small pox, syphilis, etc. Aim of the review: The review summarizes the up-to-date and comprehensive information concerning the botany, traditional use, phytochemistry and pharmacology of important drug. Kirganelia reticulata and discusses the possible future scope for future research. Material and methods: In present review covers a literature survey across from 1932 to 2012. The some information collected from published literature on species of Kirganelia reticulata (Poir.) Baill. (=Syn. Phyllanthus reticulatus Poir.) and traditional ayurvedic texts. Results: Phytochemical studies have shown the presence of many valuable compounds such as lignans, flavonoids, steroids, coumerins, triterpenes, phenols, flavonones, alkaloids are common compounds in test species. The extract and the compounds isolated from Kirganelia reticulata show a wide spectrum of Biological activities including antidiabetic, antibacterial, antioxidant, hepatoprotective, antiplasmodial, anticiceptive, analgesic and anti-inflammatory properties. Conclusion: The present review summarizes information concerning the morphology, ecology, ethanopharmacologically, phytochemistry, and traditional diseases and applications of K. reticulata. This review target at gathering the research work undertaken till date on this plant in order to provide sufficient baseline information for future works and commercial exploitation

A comparative evaluation : oral leukoplakia surgical management using diode laser, CO2 laser, and cryosurgery

The comparatively evaluate the three surgical treatment modalities namely cryosurgery, diode and CO2 laser surgery in terms of healing outcomes on the day of surgery, first and second week post operatively and recurrence at the end of 18 months was assessed. Thirty selected patients were divided randomly into three groups. Each group comprising of ten patients were subjected to one of the three modalities of treatment namely cryosurgery, diode laser or CO2 laser surgery for ablation of OL. Obtained data was analyzed using mainly using Chi-square and Anova tests. Study showed statistical significant differences (p > 0.05) for evaluation parameters like pain, edema and scar. The parameters like infection, recurrence, bleeding showed no statistical significance. Pain was significantly higher in CO2 laser surgery group as compared with diode laser group. There was no recurrence observed at the end of the 6 months follow up period in all the three study groups. Observations from the study highlights that all three surgical modalities used in this study were effective for treatment of OL, and the overall summation of the results of the study showed that laser therapy (CO2 and Diode) seems to offer better clinically significant results than cryotherapy